Inhibitors of GSK-3 and crystal structures of GSK-3β protein and protein complexes

ABSTRACT

The present invention relates to inhibitors of GSK-3 and methods for producing these inhibitors. The invention also provides pharmaceutical compositions comprising the inhibitors and methods of utilizing those compositions in the treatment and prevention of various disorders, such as diabetes and Alzheimer&#39;s disease. In addition, the invention relates to molecules or molecular complexes which comprise binding pockets of GSK-3β or its homologues. The invention relates to a computer comprising a data storage medium encoded with the structure coordinates of such binding pockets. The invention also relates to methods of using the structure coordinates to solve the structure of homologous proteins or protein complexes. The invention relates to methods of using the structure coordinates to screen for and design compounds that bind to GSK-3β protein or homologues thereof. The invention also relates to crystallizable compositions and crystals comprising GSK-3β protein or GSK-3β protein complexes.

This application is a divisional of U.S. application Ser. No.10/135,255, filed Apr. 29, 2002, now U.S. Pat. No. 7,390,808, whichclaims the benefit of U.S. Provisional Application No. 60/361,899, filedFeb. 27, 2002, U.S. Provisional Application No. 60/297,094, filed Jun.8, 2001, and U.S. Provisional Application No. 60/287,366, filed Apr. 30,2001. Each of these prior applications is hereby incorporated byreference in its entirety.

TECHNICAL FIELD OF INVENTION

The present invention relates to inhibitors of glycogen synthasekinase-3 (GSK-3), a serine/threonine protein kinase, and to methods forproducing them. The invention also provides pharmaceutical compositionscomprising the inhibitors of the invention and methods of utilizingthose compositions in the treatment and prevention of various diseasestates, such as diabetes and Alzheimer's disease. The present inventionalso relates to molecules or molecular complexes which comprise bindingpockets of GSK-3β, or its homologues. The present invention provides acomputer comprising a data storage medium encoded with the structurecoordinates of such binding pockets. This invention also relates tomethods of using the structure coordinates to solve the structure ofhomologous proteins or protein complexes. In addition, this inventionrelates to methods of using the structure coordinates to screen for anddesign compounds, including inhibitory compounds, that bind to GSK-3βprotein or homologues thereof. The invention also relates tocrystallizable compositions and crystals comprising GSK-3β protein orGSK-3β protein complexes.

BACKGROUND OF THE INVENTION

Protein kinases mediate intracellular signal transduction by affecting aphosphoryl transfer from a nucleoside triphosphate to a protein acceptorinvolved in a signaling pathway. There are a number of kinases andpathways through which extracellular and other stimuli cause a varietyof cellular responses to occur inside the cell. Examples of such stimuliinclude environmental and chemical stress signals (e.g., osmotic shock,heat shock, ultraviolet radiation, bacterial endotoxin, H₂O₂), cytokines(e.g., interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)), growthfactors (e.g., granulocyte macrophage-colony-stimulating factor(GM-CSF), and fibroblast growth factor (FGF). An extracellular stimulusmay affect one or more cellular responses related to cell growth,migration, differentiation, secretion of hormones, activation oftranscription factors, muscle contraction, glucose metabolism, controlof protein synthesis and regulation of cell cycle. Many disease statesare associated with abnormal cellular responses triggered by proteinkinase-mediated events. These diseases include autoimmune diseases,inflammatory diseases, neurological and neurodegenerative diseases,cancer, cardiovascular diseases, allergies and asthma, Alzheimer'sdisease and hormone-related diseases.

GSK-3 is a serine/threonine protein kinase and belongs to thesuperfamily of mitogen-activated protein kinases. MAP kinases areactivated by phosphorylation of threonine and/or tyrosine residues in aloop adjacent to the active site. Phosphorylation of MAP kinases iscarried out by specific kinases upstream. Activated MAP kinase thenphosphorylates the various substrates.

Mammalian cells have α and β isoforms of GSK-3 that are each encoded bydistinct genes (Coghlan et al., Chemistry & Biology, 7, pp. 793-803(2000); Kim and Kimmel, Curr. Opinion Genetics Dev., 10, pp. 508-514(2000)). The core kinase sequences have 97% similarity, but the proteinsequences deviate substantially outside the kinase core (Woodgett, J.R., EMBO J., 9, pp. 2431-8 (1990)). GSK-3α is 63 residues longer at theN-terminal end than GSK-3β, however the N-terminal phosphorylation sitein both isoforms (S21 for GSK-3α and S9 for GSK-3β) is embedded in aconserved 7 residue motif. The two isoforms are not redundant as GSK-3βdeficiency is lethal in embryogenesis due to severe liver degeneration(Hoeflich, K. P., et al., Nature, 406, pp. 86-90 (2000)).

GSK-3β has multiple phosphorylation sites. The Serine 9 and Tyrosine 216phosphorylation sites are well described in the literature.Phosphorylation of Tyrosine 216 activates GSK-3β but phosphorylation ofSerine 9 inactivates it. GSK-3β is unique among kinases in that itrequires prior phosphorylation of its substrates. GSK-3β does notphosphorylate its multiple substrates in the same manner and with thesame efficiency but has different modes of phosphorylation. Thecanonical phosphorylation sequence recognized by GSK-3β, SXXXS, containstwo serines separated by three amino acid residues. Multiple copies ofthis motif can be present in the substrate. Several protein substratessuch as glycogen synthase, eIF2b and APC, are first phosphorylated by adifferent kinase at the P+4 serine in the _(p+4)SXXXS_(p) motif beforeGSK-3β phosphorylates the serine in the P position. This is calledprimed phosphorylation, and is approximately 100 to 1000 times fasterthan the phosphorylation without priming (Thomas, G. M., et al., FEBSLett., 458, pp. 247-51 (1999)). Glycogen synthase has multiple serinesseparated by four residues (residue 640, 644, 648, and 652) and thoseserines are phosphorylated sequentially by GSK-3β from the C-terminalend, after S656 has been phosphorylated by Casein Kinase II (Woodgett,J. R. and P. Cohen, Biochim. Biophys. Acta, 788, pp. 339-47 (1984);Kuret, J. et al, Eur. J. Biochem., 151, pp. 39-48 (1985)).

Glycogen synthase kinase-3 has been implicated in various diseasesincluding diabetes, Alzheimer's disease, CNS disorders such as manicdepressive disorder and neurodegenerative diseases, and cardiomyocetehypertrophy (WO 99/65897; WO 00/38675; and Haq et al., J. Cell Biol.,151, pp. 117 (2000)). These diseases may be caused by, or result in, theabnormal operation of certain cell signaling pathways in which GSK-3plays a role. GSK-3 phosphorylates and modulates the activity of anumber of regulatory proteins. These include glycogen synthase which isthe rate limiting enzyme necessary for glycogen synthesis, themicrotubule associated protein Tau, the gene transcription factorbeta-catenin, the translation initiation factor e1F2B, as well as ATPcitrate lyase, axin, heat shock factor-1, c-Jun, c-Myc, c-Myb, CREB andCEPBa. These diverse targets implicate GSK-3 in many aspects of cellularmetabolism, proliferation, differentiation and development.

In a GSK-3 mediated pathway that is relevant for the treatment of typeII diabetes, insulin-induced signaling leads to cellular glucose uptakeand glycogen synthesis. Along this pathway, GSK-3 is a negativeregulator of the insulin-induced signal. Insulin inactivates GSK-3β viathe PKB/Akt pathway, which results in activation of glycogen synthase.(Summers, S. A., et al., J. Biol. Chem., 274, pp. 17934-40 (1999); Ross,S. E., et al., Mol. Cell. Biol., 19, pp. 8433-41 (1999)). The inhibitionof GSK-3 leads to increased glycogen synthesis and glucose uptake (Kleinet al., PNAS, 93, pp. 8455-9 (1996); Cross et al., Biochem. J., 303, pp.21-26 (1994); Cohen, Biochem. Soc. Trans., 21, pp. 555-567 (1993);Massillon et al., Biochem. J. 299, pp. 123-128 (1994)). However, in adiabetic patient where the insulin response is impaired, glycogensynthesis and glucose uptake fail to increase despite the presence ofrelatively high blood levels of insulin. This leads to abnormally highblood levels of glucose with acute and long term effects that mayultimately result in cardiovascular diseases, renal failure andblindness. In such patients, the normal insulin-induced inhibition ofGSK-3 fails to occur. It has also been reported that in patients withtype II diabetes, GSK-3 is overexpressed (WO 00/38675). Therapeuticinhibitors of GSK-3 are therefore potentially useful for treatingdiabetic patients suffering from an impaired response to insulin.

GSK-3 activity has also been associated with Alzheimer's disease. Thisdisease is characterized by the well-known β-amyloid peptide and theformation of intracellular neurofibrillary tangles. The neurofibrillarytangles contain hyperphosphorylated Tau protein where Tau isphosphorylated on abnormal sites. GSK-3 has been shown to phosphorylatethese abnormal sites in cell and animal models. Furthermore, inhibitionof GSK-3 has been shown to prevent hyperphosphorylation of Tau in cells(Lovestone et al., Current Biology, 4, pp. 1077-86 (1994); Brownlees etal., Neuroreport, 8, pp. 3251-55 (1997)). Therefore, it is believed thatGSK-3 activity may promote generation of the neurofibrillary tangles andthe progression of Alzheimer's disease.

Another substrate of GSK-3 is beta-catenin which is degraded afterphosphorylation by GSK-3. Reduced levels of beta-catenin have beenreported in schizophrenic patients and have also been associated withother diseases related to increase in neuronal cell death (Zhong et al.,Nature, 395, 698-702 (1998); Takashima et al., PNAS, 90, 7789-93 (1993);Pei et al., J. Neuropathol. Exp., 56, 70-78 (1997)).

GSK-3β is also a component of the Wnt signalling pathway. Activation ofthe Wnt pathway inhibits GSK-3β, which results in accumulation ofcytosolic β-catenin (Yost, C., et al., Cell, 93, pp. 1031-41 (1998)).The cytosolic β-catenin translocates to the cell nucleus, where itassociates with LEF/tcf and stimulates the expression of Wnt targetgenes resulting in cell proliferation (Ding, V. W., et al., J. Biol.Chem., 275, pp. 32475-81 (2000); Waltzer, L. and M. Bienz, CancerMetastasis Rev. 18, pp. 231-46 (1999); Ikeda, S., et al., EMBO J., 17,pp. 1371-84 (1998); Thomas, G. M., et al., FEBS Lett, 458, pp. 247-51(1999); Salic, A., et al., Mol. Cell., 5, pp. 523-32 (2000)). Theactivity of GSK-3β is also down regulated by 7-TM receptors thatregulate cAMP levels. cAMP-dependent protein kinase A, binds,phosphorylates and inhibits GSK-3β in response to the adenyl cyclaseactivator forskolin, or the p-adrenergic receptor activatorisoproterenol (Fang, X., et al., Proc. Natl. Acad. Sci. USA, 97, pp.11960-5 (2000)).

Small molecule inhibitors of GSK-3 have recently been reported (WO99/65897 and WO 00/38675). For many of the aforementioned diseasesassociated with abnormal GSK-3 activity, other protein kinases have alsobeen targeted for treating the same diseases. However, the variousprotein kinases often act through different biological pathways.Quinazoline derivatives have been reported recently as inhibitors of p38kinase (WO 00/12497). The compounds are reported to be useful fortreating conditions characterized by enhanced p38-α activity and/orenhanced TGF-β activity. While p38 activity has been implicated in awide variety of diseases, including diabetes, p38 kinase is not reportedto be a constituent of an insulin signaling pathway that regulatesglycogen synthesis or glucose uptake. Therefore, unlike GSK-3, p38inhibition would not be expected to enhance glycogen synthesis and/orglucose uptake.

Accordingly, there has been an interest in finding GSK-3 inhibitors thatare effective as therapeutic agents due to its important role indiabetes, Alzheimer's disease and other diseases. A challenge has beento find protein kinase inhibitors that act in a selective manner. Sincethere are numerous protein kinases that are involved in a variety ofcellular responses, non-selective inhibitors may lead to unwanted sideeffects.

In this regard, the three-dimensional structure of the kinase wouldassist in the rational design of inhibitors. Further, informationprovided by the X-ray crystal structure of GSK-3β-inhibitor complexeswould be extremely useful in iterative drug design of various GSK-3proteins. The determination of the amino acid residues in GSK-3β bindingpockets and the determination of the shape of those binding pocketswould allow one to design inhibitors that bind more favorably to thisclass of enzymes.

SUMMARY OF THE INVENTION

The present invention addresses this need by providing compounds andpharmaceutical compositions thereof that are effective as protein kinaseinhibitors, particularly as inhibitors of GSK-3. Applicants have alsoaddressed this need by providing the crystal structures of aunphosphorylated GSK-3β, a phosphorylated GSK-3β, unphosphorylatedGSK-3β-inhibitor complexes, a phosphorylated GSK-3β-inhibitor complexand a phosphorylated GSK-3β-ADP-peptide complex. Solving these crystalstructures has allowed the determination of the key structural featuresof GSK-3β, particularly the shape of its substrate and ATP-bindingpockets.

The compounds of the present invention have the general formula I:

or a pharmaceutically acceptable derivative thereof, wherein:

-   -   R₁ is selected from H, aliphatic, RC(O)—, RS(O)_(n)—, ROC(O)—,        carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or        heteroaralkyl; wherein said aliphatic, carbocyclyl,        heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl is        optionally substituted;    -   R₂ and R₃ are each independently selected from H, aliphatic,        carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl,        heteroaralkyl, —N(R)₂, —NRCOR, —NRCO₂R, —NRCO₂R, —S(O)_(n)R,        —SO₂N(R)₂, —SR, —OR, —CF₃, halo, —NO₂, —CN, —C(O)R, —CO₂R,        —OC(O)R, —CON(R)₂, or —OC(O)N(R)₂, wherein said aliphatic,        carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or        heteroaralkyl is optionally substituted; or R₂ and R₃ taken        together with the intervening atoms optionally form a five- to        nine-membered ring that is fused to the pyridazinyl ring of        formula I, said fused ring having 0-2 heteroatoms;    -   each R is independently selected from H, aliphatic, carbocyclyl,        heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl,        wherein each member of R except H is optionally substituted; and    -   n is 1 or 2;    -   provided that when R₁ is H, R₂ and R₃ are not both unsubstituted        phenyl; and when R₁ is H, R₂ and R₃ are other than H, halogen,        or an unsubstituted alkyl.

In another embodiment, the invention provides pharmaceuticalcompositions comprising a GSK-3 inhibitor of this invention. Thesecompositions may be utilized in methods for treating or preventing avariety of GSK-3 mediated disorders, such as autoimmune diseases,inflammatory diseases, metabolic, neurological and neurodegenerativediseases, cardiovascular diseases, allergy, asthma, diabetes,Alzheimer's disease, Huntington's Disease, Parkinson's Disease,AIDS-associated dementia, amyotrophic lateral sclerosis (AML, LouGehrig's Disease), multiple sclerosis (MS), schizophrenia, cardiomyocytehypertrophy, reperfusion/ischemia, and baldness.

The compositions of this invention are also useful in methods forenhancing glycogen synthesis and/or lowering blood levels of glucose andtherefore are especially useful for diabetic patients. Thesecompositions are also useful in methods for inhibiting the production ofhyperphosphorylated Tau protein, which is useful in halting or slowingthe progression of Alzheimer's disease. Another embodiment of thisinvention relates to a method for inhibiting the phosphorylation ofβ-catenin, which is useful for treating schizophrenia.

In another embodiment, the invention provides methods of synthesizingcompounds of formula I and preparing pharmaceutical compositionscomprising these compounds.

The present invention also provides molecules or molecular complexescomprising GSK-3β binding pockets, or GSK-3β-like binding pockets thathave similar three-dimensional shapes. In one embodiment, the moleculesor molecular complexes are GSK-3β proteins, protein complexes orhomologues thereof. In another embodiment, the molecules or molecularcomplexes are in crystalline form.

The invention provides crystallizable compositions and crystalcompositions comprising unphosphorylated GSK-3β, phosphorylated GSK-3βor their homologues with or without a chemical entity. The inventionalso provides a method for crystallizing a GSK-3β protein, proteincomplex, or homologues thereof.

The invention provides a data storage medium which comprises thestructure coordinates of molecules or molecular complexes of the GSK-3βbinding pockets or GSK-3β-like binding pockets. In one embodiment, thedata storage medium comprises the structure coordinates of the bindingpocket. The invention also provides a computer comprising the datastorage medium. Such storage medium when read and utilized by a computerprogrammed with appropriate software can display, on a computer screenor similar viewing device, a three-dimensional graphical representationof such binding pockets.

The invention also provides methods for designing, evaluating andidentifying compounds which bind to the molecules or molecular complexesor their binding pockets. Such compounds are potential inhibitors ofGSK-3β or its homologues.

The invention also provides a method for determining at least a portionof the three-dimensional structure of molecules or molecular complexeswhich contain at least some structurally similar features to GSK-3β,particularly GSK-3β homologues. This is achieved by using at least someof the structure coordinates obtained from the unphosphorylated orphosphorylated GSK-3β protein or protein complexes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 lists the atomic structure coordinates for unphosphorylatedGSK-3β as derived by X-ray diffraction from the crystal. FIG. 1 depictsresidues 25-119, 127-285, 301-384, 37-285 and 291-382 of SEQ ID NO:1,respectively, in order of appearance.

FIG.2 lists the atomic structure coordinates for phosphorylated GSK-3β.FIG.2 depicts SEQ ID NOS: 22-27, respectively, in order of appearance.

FIG. 3 lists the atomic structure coordinates for phosphorylatedGSK-3β-inhibitor1 complex (inhibitor 1 is4,5-Diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-ylamine). FIG. 3 depicts SEQID NOS: 28-33, respectively, in order of appearance.

FIG. 4 lists the atomic structure coordinates for unphosphorylatedGSK-3β-inhibitor 2 complex (inhibitor2 is(5-Methyl-2H-pyrazol-3-yl)-(2-pyridin-4-yl-quinazolin-4-yl)-amine)).FIG. 4 depicts SEQ ID NOS: 34-38, respectively, in order of appearance.

FIG. 5 lists the atomic structure coordinates for unphosphorylatedGSK-3β-inhibitor3 complex (inhibitor 3 is4-(5-Methyl-2-phenylamino-pyrimidin-4-yl)-1H-pyrrole-2-carboxylic acid(2-hydroxy-1-phenyl-ethyl)-amide). FIG. 5 depicts SEQ ID NOS: 39-44,respectively, in order of appearance.

FIG. 6 lists the atomic structure coordinates for unphosphorylatedGSK-3β-inhibitor4 complex (inhibitor 4 is(1H-Indazo-1-3-yl)-[2-(2-trifluoromethyl-phenyl)-quinazolin-4-yl]-amine).FIG. 6 depicts SEQ ID NOS:45-49, respectively, in order of appearance.

FIG. 7 lists the atomic structure coordinates for phosphorylated GSK-3βin complex with ADP and glycogen synthase peptide. FIG. 7 depicts SEQ IDNOS: 50 -55, respectively, in order of appearance.

FIG. 8 depicts a ribbon diagram of the overall fold of unphosphorylatedGSK-3β. The N-terminal domain corresponds to the β-strand domain andencompasses residues 25 to 138. β-strand 1 was only visible in one ofthe two molecules in the asymmetric unit and makes hydrogen bonds withβ-strand 2 although it is not part of the β-barrel. The α-helical domaincorresponds to residues 139 to 349. Key features of the kinase-fold suchas the hinge, glycine rich loop and activation-loop are indicated.

FIG. 9 depicts a superposition of unphosphorylated GSK-3β (light shade)and activated substrate-bound CDK2 (Protein Data Bank accession number1QMZ) (dark shade). The α-helical domains of GSK-3β and CDK2 weresuperimposed in QUANTA by aligning matching residues.

FIG. 10 shows a comparison between the activation loops ofunphosphorylated GSK-3β (FIG. 10A), phosphorylated GSK-3β (FIG. 10B),and phosphorylated p38γ (Protein Data Bank accession number 1CM8)(FIG.7C). In FIG. 10A, the side chains of residues R96, R180 and K205 arepointing to a phosphate ion that occupies the same position as thephosphate group of the phospho-threonine in activated p38γ (FIG. 10C),activated CDK2 and activated ERK2. In FIG. 10C, the phosphorylated Y216is flipped out of the substrate binding groove, which is similar to theposition of the phosphorylated Y185 of p38γ.

FIG. 11A shows that in the unphosphorylated GSK-3β structure, the GSK-3βsubstrate-binding groove is occupied by a phosphate ion and residues 260to 264 of a neighboring GSK-3β molecule.

FIG. 11B shows a model for the binding of the SXXXpS motif in the GSK-3βsubstrate-binding groove. The α-helical domains of GSK-3β and activatedsubstrate bound CDK2 (Protein Data Bank accession number 1QMZ) weresuperimposed to model the positioning of a primed peptide in the GSK-3βsubstrate-binding groove. The side chains of the peptide residues exceptfor the target serine S* have been removed for clarity. Thephosphorylation site S* is positioned in front of the active site. Themodel estimates how the SXXXpS motif fits in the substrate-bindinggroove with the three residues bridging the gap between the P and P+4serines. The phosphate group of the P+4 serine will occupy the sameposition as the phosphate ion found in the crystal structure.

FIG. 12 depicts a ribbon diagram of phosphorylated GSK-3β. Theactivation loop and the phosphorylated Y216 are indicated.

FIG. 13 presents the inhibitor2 bound in the active site ofunphosphorylated GSK-3β. The hinge and glycine rich loop are indicated.

FIG. 14 presents the view of inhibitor1 bound in the active site ofphosphorylated GSK-3β.

FIG. 15 presents the view of inhibitor3 bound in the active site ofunphosphorylated GSK-3β.

FIG. 16 presents the conformation of Gln185 in the active site whenbound to inhibitor4.

FIG. 17 depicts the overall structure of phosphorylated GSK-3β incomplex with ADP and glycogen synthase peptide. The N-terminal domaincorresponds to the β-strand domain and includes residues 37 to 138. Theα-helical domain of the GSK-3β kinase core corresponds to residues 139to 343. The C-terminal 34 residues are not part of the kinase core butpack against the α-helical domain. Key features of the kinase fold, suchas the glycine rich loop, the hinge and the activation loop areindicated. The ADP-Mg complex occupying the active site is shown. Theglycogen synthase peptide (residues 650 to 658) bound in the substratebinding groove is also shown here.

FIG. 18 depicts the β-strand domain rotation induced by ADP and glycogensynthase peptide. Superposition of the α-helical domains ofunphosphorylated (dark shade), phosphorylated apo-GSK3 (gray shade) andphosphorylated ADP peptide bound GSK-3β (light shade). The β-stranddomain of the phosphorylated GSK-3β-ADP-peptide complex, rotated 6.5 Åin comparison to unphosphorylated and phosphorylated apo-GSK-3. The loopbetween β-3 and α-C (residues 87 to 95) moved 13 Å to contact theglycogen synthase peptide (not shown).

FIG. 19A depicts a stereo view of the active site occupied by the ADP-Mgcomplex. The adenine base is surrounded by hydrophobic residues andmakes two hydrogen bonds with the backbone of the hinge region. Thecatalytic residues involved in the phosphate transfer to the P-siteserine surround the two non-transferable ADP phosphates.

FIG. 19B is a schematic drawing of the hydrogen bond network thatconnects the ADP-Mg complex to the P-site serine via the catalyticresidues.

FIG. 20 depicts the environment of phosphotyrosine 216 in the structureof phosphorylated GSK-3β in complex with ADP and glycogen synthasepeptide. The phosphate moiety binds two arginine side chains (Arg 220and Arg 223) resulting in charge neutralization. The arrow indicated the180 degree flip of I217 backbone carbonyl.

FIG. 21 depicts the glycogen synthase peptide in the substrate bindinggroove. The phosphoserine (pser 656) binds Arg 96, Arg 180 and Lys 205,which results in the proper alignment of the α-helical and β-stranddomains. pTyr 216 moves its side chain out of the substrate bindinggroove to make contact with the side chains of Arg 220 and Arg 223.

FIG. 22 shows a diagram of a system used to carry out the instructionsencoded by the storage medium of FIGS. 23 and 24.

FIG. 23 shows a cross section of a magnetic storage medium.

FIG. 24 shows a cross section of a optically-readable data storagemedium.

The following abbreviations are used in FIGS. 1-5:

“Atom type” refers to the element whose coordinates are measured. Thefirst letter in the column defines the element.

“Res” refers to the amino acid residue in the molecular model.

“X, Y, Z” crystallographically define the atomic position of the elementmeasured.

“B” is a thermal factor that measures movement of the atom around itsatomic center.

“Occ” is an occupancy factor that refers to the fraction of themolecules in which each atom occupies the position specified by thecoordinates. A value of “1” indicates that each atom has the sameconformation, i.e., the same position, in all molecules of the crystal.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention described herein may be more fullyunderstood, the following detailed description is set forth.

Throughout the specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or groups of integers.

The following abbreviations are used throughout the application:

A = Ala = Alanine T = Thr = Threonine V = Val = Valine C = Cys =Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = Ile = Isoleucine N =Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine F = Phe =Phenylalanine D = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu =Glutamic Acid M = Met = Methionine K = Lys = Lysine G = Gly = Glycine R= Arg = Arginine S = Ser = Serine H = His = Histidine pS =Phosphorylated Serine pTy = Phosphorylated Tyrosine

As used herein, the following definitions shall apply unless otherwiseindicated. Also, combinations of substituents or variables arepermissible only if such combinations result in stable compounds.

The term “about” when used in the context of RMSD values takes intoconsideration the standard error of the RMSD value, which is ±0.1 Å.

The term “active site” refers to the area in the protein kinase wherethe nucleotide binds. This site is located at the interface of theC-terminal α-helical and N-terminal β-strand domain, and is bordered bythe glycine rich loop and the hinge (See, Xie et al., Structure, 6, pp.983-991 (1998), incorporated herein by reference).

The term “aliphatic” refers to straight chain or branched hydrocarbonsthat are completely saturated or that contain one or more units ofunsaturation. For example, aliphatic groups include substituted orunsubstituted linear or branched alkyl, alkenyl and alkynyl groups.Unless indicated otherwise, the term “aliphatic” encompasses bothsubstituted and unsubstituted hydrocarbons. The term “alkyl”, used aloneor as part of a larger moiety, refers to both straight and branchedsaturated chains containing one to twelve carbon atoms. The terms“alkenyl” and “alkynyl”, used alone or as part of a larger moiety,encompass both straight and branched chains containing two to twelvecarbon atoms and at least one unit of unsaturation. An alkenyl groupcontains at least one carbon-carbon double bond and an alkynyl groupcontains at least one carbon-carbon triple bond.

The term “correspond to” or “corresponding amino acids” when used in thecontext of amino acid residues that correspond to GSK-3β amino acidsrefers to particular amino acids or analogues thereof in a protein thatcorrespond to amino acids in the GSK-3β protein. The corresponding aminoacid may be an identical, mutated, chemically modified, conserved,conservatively substituted, functionally equivalent or homologous aminoacid when compared to the GSK-3β amino acid to which it corresponds.

Methods for identifying a corresponding amino acid are known in the artand are based upon sequence, structural alignment, its functionalposition or a combination thereof as compared to the GSK-3β protein. Forexample, corresponding amino acids may be identified by superimposingthe backbone atoms of the amino acids in GSK-3β and the protein usingwell known software applications, such as QUANTA (Molecular Simulations,Inc., San Diego, Calif. ©2000). The corresponding amino acids may alsobe identified using sequence alignment programs such as the “bestfit”program available from the Genetics Computer Group which uses the localhomology algorithm described by Smith and Waterman in Advances inApplied Mathematics 2, 482 (1981), which is incorporated herein byreference.

The term “aryl”, alone or in combination with other terms, refers tomonocyclic or polycyclic aromatic carbon ring systems having five tofourteen members. Examples of aryl groups include, but are not limitedto, phenyl (Ph), 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl.The term “aralkyl” refers to an alkyl group substituted by an aryl. Alsoexplicitly included within the scope of the term “aralkyl” are alkenylor alkynyl groups substituted by an aryl. Examples of aralkyl groupsinclude benzyl and phenethyl. The term “aryl”, “aryl group” or “arylring” also refers to rings that are optionally substituted, unlessotherwise indicated.

The term “associating with” refers to a condition of proximity between achemical entity or compound, or portions thereof, and a binding pocketor binding site on a protein. The association may benon-covalent—wherein the juxtaposition is energetically favored byhydrogen bonding or van der Waals or electrostatic interactions—or itmay be covalent.

The term “ATP analogue” refers to a compound derived fromAdenosine-5′-triphosphate(ATP). The analogue can be ADP, ornon-hydralysable, for example, Adenylyl Imidodiphosphate (AMPPNP).AMPPNP can be in complex with Magnesium or Manganese ions.

The term “binding pocket” refers to a region of a molecule or molecularcomplex, that, as a result of its shape, favorably associates withanother chemical entity or compound.

The term “biological sample” includes, without limitation, cell culturesor extracts thereof; biopsied material obtained from a mammal orextracts thereof; and blood, saliva, urine, feces, semen, tears, orother body fluids or extracts thereof.

The term “carbocylyl” or “carbocyclic”, alone or in combination with anyother term, refers to monocyclic or polycyclic non-aromatic carbon ringsystems, which may contain a specified number of carbon atoms,preferably from 3 to 12 carbon atoms, which are completely saturated orwhich contain one or more units of unsaturation. A carbocyclic ringsystem may be monocyclic, bicyclic or tricyclic. A carbocylyl ring maybe fused to another ring, such as an aryl ring or another carbocyclicring. Examples of carbocyclic rings could include cyclohexyl,cyclopentyl, cyclobutyl, cyclopropyl, cyclohexenyl, cyclopentenyl,indanyl, tetrahydronaphthyl and the like. The term “carbocyclic” or“carbocylyl”, whether saturated or unsaturated, also refers to ringsthat are optionally substituted unless indicated. The term “carbocyclic”or “carbocylyl” also encompasses hybrids of aliphatic and carbocyclicgroups, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl and(cycloalkyl)alkenyl.

The term “chemically feasible or stable” refers to a compound structurethat is sufficiently stable to allow manufacture and administration to apatient by methods known in the art. Typically, such compounds arestable at a temperature of 40° C. or less, in the absence of moisture orother chemically reactive conditions, for at least one week.

The term “chemical entity” refers to chemical compounds, complexes of atleast two chemical compounds, and fragments of such compounds orcomplexes. The chemical entity can be, for example, a ligand, asubstrate, nucleotide triphosphate, a nucleotide, an agonist,antagonist, inhibitor, antibody, peptide, protein or drug. In oneembodiment, the chemical entity is selected from the group consisting ofan ATP and an inhibitor for the active site. In one embodiment, theinhibitor is 4,5-Diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-ylamine,(5-Methyl-2H-pyrazol-3-yl)-(2-pyridin-4-yl-quinazolin-4-yl)-amine,4-(5-Methyl-2-phenylamino-pyrimidin-4-yl)-1H-pyrrole-2-carboxylic acid(2-hydroxy-1-phenyl-ethyl)-amide,(1H-Indazol-3-yl)-[2-(2-trifluoromethyl-phenyl)-quinazolin-4-yl]-amineand an ATP analogue such as MgAMP-PNP (adenylyl imidodiphosphate) orADP. In one embodiment, the chemical entity is selected from the groupconsisting of a peptide substrate or inhibitor for the substrate bindinggroove.

The term “crystallization solution” refers to a solution that promotescrystallization of macromolecules. The crystallization solution maycontain a precipitant, a buffer, salt, stabilizer, a polyionic agent, adetergent, a lanthanide ion or reducing agent. One of ordinary skilledin the art may adjust the components of the crystallization solution tofind a condition suitable for the macromolecule of interest.

The term “conservative substitutions” refers to residues that arephysically or functionally similar to the corresponding referenceresidues. That is, a conservative substitution and its reference residuehave similar size, shape, electric charge, chemical properties includingthe ability to form covalent or hydrogen bonds, or the like. Preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al., Atlas of Protein Sequenceand Structure, 5, pp. 345-352 (1978 & Supp.), which is incorporatedherein by reference. Examples of conservative substitutions aresubstitutions including but not limited to the following groups: (a)valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine;(d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine,threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,tyrosine.

The term “complex” refers to a protein associated with a chemicalentity.

The term “domain” refers to a structural unit of the GSK-3β protein orhomologue. The domain can comprise a binding pocket, or a sequence orstructural motif. In GSK-3β, the protein is separated into two domains,the N-terminal domain which is predominantly β strands and theC-terminal domain which is predominantly a helical.

The term “generating a three-dimensional structure” refers to plottingthe structure coordinates in three-dimensional space. This can beachieved through commercially available software. The three-dimensionalstructure may be used to perform computer modeling, fitting operations,or displayed as a three-dimensional graphical representation.

The term “GSK-3β inhibitor-binding pocket” or “GSK-3β ATP-bindingpocket” refers to a binding pocket of a molecule or molecular complexdefined by the structure coordinates of a certain set of amino acidresidues present in the GSK-3β structure, as described below. Thisbinding pocket is in an area in the GSK-3β protein where the ATP orinhibitor for the active site binds.

The term “GSK-3β-like” refers to all or a portion of a molecule ormolecular complex that has a commonality of shape to all or a portion ofthe GSK-3β protein. For example, in the GSK-3β-like inhibitor bindingpocket, the commonality of shape is defined by a root mean squaredeviation of the structure coordinates of the backbone atoms between theamino acids in the GSK-3β-like inhibitor-binding pocket and the GSK-3βamino acids in the GSK-3β inhibitor-binding pocket (as set forth in anyone of FIG. 1-7). Depending on the set of GSK-3β amino acids that definethe GSK-3β inhibitor-binding pocket, one skilled in the art would beable to locate the corresponding amino acids that define a GSK-3β-likeinhibitor-binding pocket in a protein based on sequence or structuralhomology.

The term “GSK-3-mediated condition” or “state” refers to any disease orother deleterious condition or state in which GSK-3, in particularGSK-3, is known to play a role. Such diseases or conditions include,without limitation, diabetes, Alzheimer's disease, Huntington's Disease,Parkinson's Disease, AIDS-associated dementia, amyotrophic lateralsclerosis (AML), multiple sclerosis (MS), schizophrenia, cardiomycetehypertrophy, reperfusion/ischemia, and baldness.

The term “halogen” or “halo” means F, Cl, Br, or I.

The term “heteroatom” means N, O, or S and shall include any oxidizedform of nitrogen and sulfur, such as N(O), S(O), S(O)₂ and thequaternized form of any basic nitrogen.

The term “heterocyclic” or “heterocyclyl” refers to non-aromaticsaturated or unsaturated monocyclic or polycyclic ring systemscontaining one or more heteroatoms and with a ring size of three tofourteen. One having ordinary skill in the art will recognize that themaximum number of heteroatoms in a stable, chemically feasibleheterocyclic ring is determined by the size of the ring, degree ofunsaturation, and valence. In general, a heterocyclic ring may have oneto four heteroatoms so long as the heterocyclic ring is chemicallyfeasible and stable and may be fused to another ring, such as acarbocyclic, aryl or heteroaryl ring, or to another heterocyclic ring. Aheterocyclic ring system may be monocyclic, bicyclic or tricyclic. Alsoincluded within the scope of within the scope of the term “heterocyclic”or “heterocyclyl”, as used herein, is a group in which one or morecarbocyclic rings are fused to a heteroaryl.

Examples of heterocyclic rings include, but are not limited to,3-1H-benzimidazol-2-one, 3-1H-alkyl-benzimidazol-2-one,2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl,3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino,2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl,2-pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl,1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl,4-thiazolidinyl, diazolonyl, N-substituted diazolonyl, 1-phthalimidinyl,benzoxane, benzotriazol-1-yl, benzopyrrolidine, benzopiperidine,benzoxolane, benzothiolane, benzothiane, aziranyl, oxiranyl, azetidinyl,pyrrolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, pyrazolinyl,pyrazolidinyl, pyranyl, dioxanyl, dithianyl, trithianyl, quinuclidinyl,oxepanyl, and thiepanyl. The term “heterocyclic” ring, whether saturatedor unsaturated, also refers to rings that are optionally substituted,unless otherwise indicated.

The term “heteroaryl”, alone or in combination with any other term,refers to monocyclic or polycyclic aromatic ring systems having five tofourteen members and one or more heteroatoms. One having ordinary skillin the art will recognize that the maximum number of heteroatoms in astable, chemically feasible heteroaryl ring is determined by the size ofthe ring and valence.

The term “heteroaralkyl” refers to an alkyl group substituted by aheteroaryl. Also explicitly included within the scope of the term“heteroaralkyl”, are alkenyl or alkynyl groups substituted by aheteroaryl. In general, a heteroaryl ring may have one to fourheteroatoms. Heteroaryl groups include, without limitation, 2-furanyl,3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl,2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl,3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 5-tetrazolyl,2-triazolyl, 5-triazolyl, 2-thienyl, and 3-thienyl. The term “heteroarylring”, “heteroaryl group”, or “heteroaralkyl” also refers to rings thatare optionally substituted.

Examples of fused polycyclic heteroaryl and aryl ring systems in which acarbocyclic aromatic ring or heteroaryl ring is fused to one or moreother rings include, tetrahydronaphthyl, benzimidazolyl, benzothienyl,benzofuranyl, indolyl, quinolinyl, benzothiazolyl, benzoxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisoxazolyl,and the like.

An aryl, aralkyl, heteroaryl, or heteroaralkyl group may contain one ormore independently selected substituents. Examples of suitablesubstituents on the unsaturated carbon atom of an aryl or heteroarylgroup include halogen, CF₃, —R′, —OR′, —OH, —SH, —SR′, protected OH(such as acyloxy), —NO₂, —CN, —NH₂, —NHR′, —N(R′)₂, —NHCOR′, —NHCONH₂,—NHCONHR′, —NHCON(R′)₂, —NRCOR′, —NHCO₂H, —NHCO₂R′, —CO₂R′, —CO₂H,—COR′, —CONH₂, —CONHR′, —CON(R′)₂, —S(O)₂H, —S(O)₂R′, —SO₂NH₂, —S(O)H,—S(O)R′, —SO₂NHR′, —SO₂N(R′)₂, —NHS(O)₂H, or —NHS(O)₂R′, where R′ isselected from H, aliphatic, carbocyclyl, heterocyclyl, aryl, aralkyl,heteroaryl, or heteroaralkyl and each R′ is optionally substituted withhalogen, nitro, cyano, amino, —NH— (unsubstituted aliphatic),—N-(unsubstituted aliphatic)₂, carboxy, carbamoyl, hydroxy, —O—(unsubstituted aliphatic), —SH, —S— (unsubstituted aliphatic), CF₃,—SO₂NH₂, unsubstituted aliphatic, unsubstituted carbocyclyl,unsubstituted heterocyclyl, unsubstituted aryl, unsubstituted aralkyl,unsubstituted heteroaryl, or unsubstituted heteroaralkyl.

An aliphatic group or a non-aromatic heterocyclic ring may contain oneor more substituents. Examples of suitable substituents on the saturatedcarbon of an aliphatic group or of a non-aromatic heterocyclic ringinclude those listed above for the unsaturated carbon as well as thefollowing: ═O, ═S; ═NNHR′, ═NN(R′)₂, ═N—OR′, ═NNHCOR′, ═NNHCO₂R′,═NNHSO₂R′, ═N—CN, or ═NR′, wherein R′ is as defined above. Guided bythis specification, the selection of suitable substituents is within theknowledge of one skilled in the art.

A substitutable nitrogen on a heteroaryl or a non-aromatic heterocyclicring is optionally substituted. Suitable substituents on the nitrogeninclude R″, COR″, S(O)₂R″, and CO₂R″, where R″ is H, an aliphatic groupor a substituted aliphatic group.

The term “motif” refers to a group of amino acids in the protein thatdefines a structural compartment or carries out a function in theprotein, for example, catalysis, structural stabilization orphosphorylation. The motif may be conserved in sequence, structure andfunction when. The motif can be contiguous in primary sequence orthree-dimensional space. Examples of a motif include but are not limitedto SXXXS motif, phosphorylation lip or activation loop, the glycine-richphosphate anchor loop, the catalytic loop, the DFG loop and the APEmotif (See, Xie et al., Structure, 6, pp. 983-991 (1998)).

The term “homologue of GSK-3β” or “GSK-3β homologue” refers to amolecule that is homologous to GSK-3β by structure or sequence, butretains the kinase activity of GSK-3. In one embodiment, the homologuehas at least 80%, 90% or 95% sequence homology to GSK-3β. The homologuecan be GSK-3α, GSK-3β from another species, with conservativesubstitutions, conservative additions or deletions thereof; human GSK-3βwith conservative substitutions, conservative additions or deletions.For example, the GSK-3β can be full length protein (amino acids 1-420 ofSEQ ID NO: 1); a truncated protein with amino acids 7-420, 25-381,37-381 of SEQ ID NO: 1; the full length protein with conservativesubstitutions; the truncated protein with conservative mutations.

The term “part of a binding pocket” refers to less than all of the aminoacid residues that define the binding pocket. The structure coordinatesof residues that constitute part of a binding pocket may be specific fordefining the chemical environment of the binding pocket, or useful indesigning fragments of an inhibitor that may interact with thoseresidues. For example, the portion of residues may be key residues thatplay a role in ligand binding, or may be residues that are spatiallyrelated and define a three-dimensional compartment of the bindingpocket. The residues may be contiguous or non-contiguous in primarysequence.

In one embodiment, part of the binding pocket is at least two amino acidresidues. In one embodiment, part of the inhibitor-binding pocket isGSK-3β amino acids D133 and V135. In another embodiment, part of theinhibitor-binding pocket is GSK-3β amino acids K85, L132, D133 and V135.In another embodiment, part of the inhibitor-binding pocket is GSK-3βamino acids K85, M101, V110, L130 and L132. In another embodiment, partof the inhibitor-binding pocket is GSK-3β amino acids I62, V135, P136,T138 and L188. In another embodiment, part of the inhibitor-bindingpocket is GSK-3β amino acids V70, V110, L188 and C199. In anotherembodiment, part of the inhibitor-binding pocket is GSK-3β amino acidsF67, V70, Q185 and C199. In one embodiment, part of a substrate bindingpocket is GSK-3β amino acids D90, K91, R92, F93, K94. In anotherembodiment, part of the substrate binding pocket is GSK-3β amino acidsR96, R180, K205, N213 and Y234. In another embodiment, part of thesubstrate binding pocket is GSK-3β amino acids R96, R180 and K205. Inanother embodiment, part of the substrate binding pocket is GSK-3β aminoacids S66, F67 and F93. In another embodiment, part of the substratebinding pocket is GSK-3β amino acids Y216, 1217, C218, S219, R220 andR223.

The term “part of a GSK-3β protein” refers to less than all of the aminoacid residues of a GSK-3β protein. In one embodiment, part of a GSK-3βprotein defines the binding pockets, domains or motifs of the protein.The structure coordinates of residues that constitute part of a GSK-3βprotein may be specific for defining the chemical environment of theprotein, or useful in designing fragments of an inhibitor that mayinteract with those residues. The portion of residues may also beresidues that are spatially related and define a three-dimensionalcompartment of a binding pocket, motif or domain. The residues maybecontiguous or non-contiguous in primary sequence. For example, theportion of residues may be key residues that play a role in ligand orsubstrate binding, catalysis or structural stabilization.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that may beadministered to a patient, together with a compound of this invention,and which does not destroy the pharmacological activity thereof.

The term “patient” includes human and veterinary subjects.

The term “peptide comprising a phosphorylation sequence” refers to apeptide comprising the ZXXXY motif Z can be serine or threonine. Y canbe serine, threonine or valine. X can be any amino acid residue. Z or Ycan be phosphorylated or unphosphorylated. The phosphorylation of Yfacilitates the phosphorylation of X by GSK-3β. Examples of peptidesubstrates comprising a phosphorylation sequence include but are notlimited to, ASVPPS (SEQ ID NO: 2), PSPSLS (SEQ ID NO: 3), LSRHSS (SEQ IDNO: 4), SSPHQS (SEQ ID NO: 5), DSRAGS (SEQ ID NO: 6), LSRRPS (SEQ ID NO:7), PTPPPT (SEQ ID NO: 8), PTPVPS (SEQ ID NO: 9), KSPVVS (SEQ ID NO:10), VSGDTS (SEQ ID NO: 11), QSYLDS (SEQ ID NO: 12), DSGIHS (SEQ ID NO:13), HSGATT (SEQ ID NO: 14), TTTAPS (SEQ ID NO: 15), TSANDS (SEQ ID NO:16), DSEQQS (SEQ ID NO: 17), SSPLPS (SEQ ID NO: 18), PSSPLS ((SEQ ID NO:19), and CTPTDV (SEQ ID NO: 20).

The term “pharmaceutically acceptable derivative” or “prodrug” means anypharmaceutically acceptable salt, ester, salt of an ester or otherderivative of a compound of this invention which, upon administration toa recipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof. Particularly favored derivatives or prodrugs are thosethat increase the bioavailability of the compounds of this inventionwhen such compounds are administered to a patient (e.g., by allowing anorally administered compound to be more readily absorbed into the blood)or which enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies.

Pharmaceutically acceptable prodrugs of the compounds of this inventioninclude, without limitation, esters, amino acid esters, phosphateesters, metal salts and sulfonate esters.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN⁺(C₁₋₄ alkyl)₄ salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

The term “protein kinase-mediated condition” or “state” refers to anydisease or other deleterious condition or state in which a proteinkinase is known to play a role. Such conditions include, withoutlimitation, autoimmune diseases, inflammatory diseases, metabolic,neurological and neurodegenerative diseases, cardiovasclular diseases,allergy and asthma.

The term “root mean square deviation” or “RMSD” refers to the squareroot of the arithmetic mean of the squares of the deviations from themean. It is a way to express the deviation or variation from a trend orobject. For purposes of this invention, the “root mean square deviation”defines the variation in the backbone of a protein from the backbone ofGSK-3β or a binding pocket portion thereof, as defined by the structurecoordinates of GSK-3β described herein. It would be readily apparent tothose skilled in the art that the calculation of RMSD involves standarderror.

The term “soaked” refers to a process in which the crystal istransferred to a solution containing the compound of interest.

The term “structure coordinates” refers to Cartesian coordinates derivedfrom mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays by the atoms (scatteringcenters) of a protein or protein complex in crystal form. Thediffraction data are used to calculate an electron density map of therepeating unit of the crystal. The electron density maps are then usedto establish the positions of the individual atoms of the enzyme orenzyme complex.

The term “substantially all of a GSK-3β binding pocket” or“substantially all of a GSK-3β protein” refers to all or almost all ofthe amino acids in the GSK-3β binding pocket or protein. For example,substantially all of a GSK-3β binding pocket can be 100%, 95%, 90%, 80%,70% of the residues defining the GSK-3β binding pocket or protein.

The term “substrate binding groove” refers to an area in a proteinkinase where the substrate binds. The substrate binding groove islocated at the interface of the β-strand and α-helical domain, andpositioned between the activation loop and β-strand domain. Examples ofsubstrates include but are not limited to glycogen synthase, β-catenin,elongation initiation factor 2B ε subunit, cAMP-responsive elementbinding protein, CCAAT/enhancer binding protein α, microtuble associatedprotein Tau, axin, Dd-STATa and Cyclin D1.

The term “substrate binding pocket” refers to a binding pocket of amolecule or molecular complex defined by the structure coordinates of acertain set of amino acid residues present in the GSK-3β structure, asdescribed below. This binding pocket is in an area in the GSK-3β proteinwhere the substrate binding groove is located.

The term “sufficiently homologous to GSK-3β” refers to a protein thathas a sequence homology of at least 20% compared to GSK-3β protein. Inone embodiment, the sequence homology is at least 40%.

Inhibitors of GSK-3

One object of the instant invention is to provide compounds havingformula (I):

or a pharmaceutically acceptable derivative thereof, wherein:

-   R₁ is selected from H, aliphatic, RC(O)—, RS(O)_(n)—, ROC(O)—,    carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or    heteroaralkyl; wherein said aliphatic, carbocyclyl, heterocyclyl,    aryl, aralkyl, heteroaryl, or heteroaralkyl is optionally    substituted;-   R₂ and R₃ are each independently selected from H, aliphatic,    carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,    —N(R)₂, —NRCOR, —NRCO₂R, —NRSO₂R, —S(O)_(n)R, —SO₂N(R)₂, —SR, —OR,    —CF₃, halo, —NO₂, —CN, —C(O)R, —CO₂R, —OC(O)R, —CON(R)₂, or    —OC(O)N(R)₂, wherein said aliphatic, carbocyclyl, heterocyclyl,    aryl, aralkyl, heteroaryl, or heteroaralkyl is optionally    substituted; or R₂ and R₃ taken together with the intervening atoms    optionally form a five- to nine-membered ring that is fused to the    pyridazinyl ring of formula I, said fused ring having 0-2    heteroatoms;-   each R is independently selected from H, aliphatic, carbocyclyl,    heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, wherein    each member of R except H is optionally substituted; and-   n is 1 or 2;-   provided that when R₁ is H, R₂ and R₃ are not both unsubstituted    phenyl; and when R₁ is H, R₂ and R₃ are other than H, halogen, or an    unsubstituted alkyl.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³C— or ¹⁴C-enriched carbonare within the scope of this invention.

In a preferred embodiment of the invention, R₁ is H, RC(O)—, or aralkyl,wherein R is as defined above. In a more preferred embodiment, R₁ is H,aliphatic-C(O)—, aryl-C(O)—, or aralkyl. In an even more preferredembodiment, R₁ is H, CH₃C(O)—, PhC(O)—, or PhCH₂—.

In another preferred embodiment, R₂ and R₃ are independently H, aryl orheteroaryl. In another preferred embodiment, R₂ and R₃ are independentlyH, aryl, carbocyclyl, heterocyclyl, or heteroaryl. In anotherembodiment, R₂ and R₃ are independently aryl, carbocyclyl, heterocyclyl,or heteroaryl. Pref erably, R₂ and R₃ are independently H, phenyl,naphthyl, pyridyl, thienyl, furanyl, pyrimidinyl, benzodioxolyl, orcyclohexyl, any of which except H is optionally substituted. Morepreferably, R₂ and R₃ are independently phenyl, naphthyl, pyridyl,thienyl, furanyl, pyrimidinyl, benzodioxolyl, or cyclohexyl, any ofwhich is optionally substituted. Even more preferably, the substituentson phenyl, naphthyl, pyridyl, thienyl, furanyl, pyrimidinyl,benzodioxolyl, or cyclohexyl are selected from halo, alkyl, —CN, —NO₂,—SO₂NH₂, —SO₂NH-(alkyl), —SO₂N(alkyl)₂, —O-alkyl, —NH₂, —N-alkyl,—N-(alkyl)₂, —CONH₂, —CONH(alkyl), —CONH(alkyl)₂, —O-phenyl, or—S-alkyl.

In another preferred embodiment, when R₁ is a large group, R₂ is a smallgroup. A small group refers to hydrogen or a moiety that contains 3carbons or less, such as methyl, ethyl, or propyl. A large group refersto a moiety that contains 4 or more carbons.

In another preferred embodiment, R₁ is H, and R₂ and R₃ areindependently H or an optionally substituted phenyl.

A more preferred embodiment of the invention is shown in formula Ia:

(Ia), wherein R₄ is halo. In an even more preferred embodiment, R₄ is F.

Representative examples of compounds of the present invention are shownbelow in Table 1.

TABLE 1

Compound No. R₁ R₂ R₃ 1 H

2 H

3 H

4 H

5 H

6 H

7 H

8 H

9

10

11 H

12 H

13 H

14 H

15 H

16 H

17 H

18 H

19 H

20 H

21 H

22 H

23 H

24 H

25

26 H

27 H

28 H

29 H

30 H

31 H

32 H

H 33 H H

35 H

36 H

37 H

38 H

39 H

40 H

41 H

42 H

43 H

44 H

45 H

46 H

47 H

48 H

49 H

50 H

51 H

52 H

53 H

54 H

55 H

56 H

57 H

58 H

59 H

60 H

61 H

62 H

63 H

64 H

65 H

66 H

67 H

68 H

69 H

70 H

71 H

72 H

73 H

74 H

75 H

76 H

77 H

78 H

79 H

80 H

81 H

82 H

83 H

84 H

85 H

86 H

87 H

88 H

89 H

90 H

91 H

92 H

93 H

94 H

95 H

96 H

97 H

98 H

99 H

100 H

101 H

102 H

In a more preferred embodiment, the compound of the present invention is3-amino-4-phenyl-5-(3-fluorophenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 8).

Methods for Producing GSK-3 Inhibitors

The compounds of this invention generally may be prepared from knownstarting materials, following methods known to those skilled in the artfor analogous compounds, as illustrated by general Scheme I and thesynthetic examples described below. References that may be useful inmaking the present compounds include El-Gendy, A. M. et al., Asian J.Chem., 1, 376 (1989); Deeb, A. and Said, S. A., Collect. Czech. Chem.Comm., 50, 2795 (1990); and Shalaby, A. A. J. Prakt. Chemie, 332, 104(1990).

Scheme I shows a general approach for making the present compounds. Theunsymmetrical diaryl keto hydrazones (1) were prepared from thecorresponding substituted deoxybenzoins as described in U.S. Pat. No.4,009,022, incorporated herein by reference. The substituteddeoxybenzoins were readily synthesized following methods known in theart, for instance, those described in Hill, D. T. et al, J. Het. Chem.,28, 1181 (1991); Rieke, R. D. et al, J. Org. Chem., 56, 1445 (1991);Fujisowa, T. et al, Chem. Lett., 1135 (1981); and Iyoda, M. et al, Tet.Lett., 26, 4777 (1985). To an ethanol solution of diaryl keto hydrazone(1), ethyl cyanoacetate (excess) and sodium ethoxide in tetrahydrofuran(THF) were added. The mixture was refluxed for 6 hours. After cooling,the solvent was removed under vacuum and the residue was taken up indichloromethane (CH₂Cl₂), washed with 0.1 M HCl and water and dried withsodium sulfate. After filtering, the solvent was removed under vacuumand the product, 4-cyano-5,6-diaryl 2(1H) pyridazinone (2) was purifiedby chromatography on silica gel (5:95 methanol/dichloro-methane).

Purified 4-cyano-5,6-diaryl 2(1H) pyridazinone (2) was added to POCl₃and heated to 100° C. for 5-6 hours. After cooling, the reaction mixturewas poured onto ice and stirred for one hour. The resultant3-chloro-4-cyano-5,6-diaryl pyridazine (3) was filtered off, washed withwater, air dried and used in the next step without further purification.Purified 3-chloro-4-cyano-5,6-diaryl pyridazine (3) was further refluxedwith 2 equivalents of anhydrous hydrazine in ethanol for several hours.Upon cooling, the product Ib would sometimes precipitate out, in whichcase compound Ib was purified by recrystallizing from ethanol. Otherwisepurification of Ib was achieved by chromatography on silica gel (5:95methanol/dichloromethane).

One having ordinary skill in that art may synthesize other compounds ofthis invention following the teachings of the specification usingreagents that are readily synthesized or commercially available.

The present invention provides detailed methods of producingrepresentative compounds of the present invention as described inExamples 1-17 below.

The activity of the compounds as protein kinase inhibitors, for example,as GSK-3 inhibitors, may be assayed in vitro, in vivo or in a cell line.In vitro assays include assays that determine inhibition of either thephosphorylation activity or ATPase activity of activated GSK-3.Alternate in vitro assays quantitate the ability of the inhibitor tobind to GSK-3. Inhibitor binding may be measured by radiolabelling theinhibitor prior to binding, isolating the inhibitor/GSK-3 complex anddetermining the amount of radiolabel bound. Alternatively, inhibitorbinding may be determined by running a competition experiment where newinhibitors are incubated with GSK-3 bound to known radioligands.

Pharmaceutical Compositions

According to another embodiment of the invention, the protein kinaseinhibitors, particularly GSK-3 inhibitors, or derivatives/salts thereofmay be formulated into compositions. In a preferred embodiment, thecomposition is a pharmaceutical composition. In one embodiment, thecomposition comprises an amount of the protein kinase inhibitoreffective to inhibit GSK-3 in a biological sample or in a patient. Inanother embodiement, the pharmaceutical compositions, which comprise anamount of the protein kinase inhibitor effective to treat or prevent aGSK-3-mediated condition and a pharmaceutically acceptable carrier,adjuvant, or vehicle, may be formulated for administration to a patient.

The amount effective to inhibit GSK-3 is one that inhibits the kinaseactivity of GSK-3 at least 50%, more preferably at least 60% or 70%,even more preferably at least 80% or 90%, and most preferably at least95%, where compared to the GSK-3 activity of the enzyme in the absenceof an inhibitor. Any method may be used to determine inhibition. See,e.g., Example 18.

Pharmaceutically acceptable carriers that may be used in thesepharmaceutical compositions include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,favoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

In addition to the compounds of this invention, pharmaceuticallyacceptable derivatives or prodrugs of the compounds of this inventionmay also be employed in compositions to treat or prevent theabove-identified diseases or disorders.

The amount of the protein kinase inhibitor that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the patient treated and the particular mode of administration.Preferably, the compositions should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of the inhibitor can beadministered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of inhibitor will also depend upon the particular compound in thecomposition.

Method of Treatment and Prevention of Disease

One aspect of this invention relates to a method for treating a diseasestate in patients that is alleviated by treatment with a GSK-3inhibitor, which method comprises administering to a patient in need ofsuch a treatment a therapeutically effective amount of a compound offormula I or a pharmaceutically acceptable derivative thereof.

Another aspect of this invention relates to a method of inhibiting GSK-3activity in a patient, comprising administering to the patient acomposition comprising a compound of formula I or a pharmaceuticallyacceptable derivative thereof. Another method relates to enhancingglycogen synthesis and/or lowering blood levels of glucose in a patientin need thereof, which method comprises administering to the patient atherapeutically effective amount of a compound of formula I or apharmaceutically acceptable derivative thereof. This method isespecially useful for diabetic patients. Another method relates toinhibiting the production of hyperphosphorylated Tau protein, which isuseful in halting or slowing the progression of Alzheimer's disease.Another method relates to inhibiting the phosphorylation of β-catenin,which is useful for treating schizophrenia.

Depending upon the particular protein kinase-mediated condition to betreated or prevented, additional drugs, which are normally administeredto treat or prevent that condition, may be administered together withthe inhibitors of this invention. For example, in the treatment ofdiabetes, other anti-diabetic agents may be combined with the GSK-3inhibitors of this invention to treat diabetes. These agents include,without limitation, insulin, in injectable or inhalation form,glitazones, and sulfonyl ureas.

Those additional agents may be administered separately from the proteinkinase inhibitor-containing composition, as part of a multiple dosageregimen. Alternatively, those agents may be part of a single dosageform, mixed together with the protein kinase inhibitor of this inventionin a single composition.

Another method of this invention relates to inhibiting GSK-3 activity ina biological sample, which method comprises contacting the biologicalsample with the GSK-3 inhibitor of formula I or a pharmaceuticallyacceptable derivative or prodrug thereof, or a pharmaceuticalcomposition thereof, in an amount effective to inhibit GSK-3.

Crystallizable Compositions and Crystals of GSK-3β Protein and ProteinComplexes

According to one embodiment, the invention provides a crystallizablecomposition comprising unphosphorylated GSK-3β protein or its homologueand phosphate ions. In one embodiment, the crystallizable compositionfurther comprises between about 5 to 25% v/v of precipitant polyethyleneglycol, a buffer that maintains pH between about 4.0 and 8.0; andoptionally, a reducing agent of 1-20 mM. In one embodiment, thecrystallizable composition comprises unphosphorylated GSK-3β protein,15% PEG 3350, 50 mM Na/KPO₄ at pH 4.1 and 10 mM DTT.

In another embodiment, the invention provides a crystallizablecomposition comprising phosphorylated GSK-3β protein or its homologue.In one embodiment, the crystallizable composition further comprisesbetween about 5-25% v/v polyethylene glycol, a buffer that maintains pHbetween about 6.0 and 8.5, and 1-10% dimethyl sulfoxide(DMSO). In oneembodiment, the crystallizable composition comprises phosphorylatedGSK-3β protein, between about 7-10% PEG 3350, 100 mM Tris HCl and 5%DMSO.

In another embodiment, the invention provides a crystallizablecomposition comprising GSK-3β protein or its homologue and a chemicalentity. The GSK-3β protein may be phosphorylated or unphosphorylated. Inone embodiment, the chemical entity is selected from the groupconsisting of an inhibitor for the active site, a nucleotidetriphosphate, an ATP, a substrate or inhibitor for the substrate bindinggroove, or a peptide comprising a phosphorylation sequence. In oneembodiment, the inhibitor for the active site is selected from the groupconsisting of 4,5-Diphenyl-1H-pyrazolo [3,4-c]pyridazin-3 -ylamine;(5-Methyl-2H-pyrazo 1-3 -yl)-(2-pyridin-4-yl-quinazolin-4-yl)-amine;4-(5-Methyl-2-phenylamino-pyrimidin-4-yl)- 1H-pyrrole-2-carboxylic acid(2-hydroxy-1 -phenyl-ethyl)-amide; (1H-Indazol-3-yl)-[2-(2-trifluoromethyl-phenyl)-quinazolin-4-yl]-amine and an ATPanalogue. In one embodiment, the crystallizable composition furthercomprises between about 5-25% v/v polyethylene glycol, and between about0.1-1 M ammonium fluoride, ammonium formate, potassium formate orpotassium fluoride. In one embodiment, the peptide comprising aphosphorylation sequence is HSSPHQpSEDEEE (SEQ ID NO: 21). In anotherembodiment, the crystallizable composition comprises phosphorylatedGSK-3βprotein, HSSPHQpSEDEEE (SEQ ID NO: 21),between about 10-15% v/vpolyethylene glycol, and 50 mM ammonium fluoride.

In one embodiment, the GSK-3β protein or its homologue is preferably85-100% pure prior to forming the crystallizable composition.

According to another embodiment, the invention provides a crystalcomposition comprising unphosphorylated GSK-3β protein or its homologueand phosphate ions. In another embodiment, the invention provides acrystal composition comprising unphosphorylated GSK-3β protein or itshomologue and a chemical entity. Preferably, the chemical entity is aninhibitor for the active site, an ATP analogue or nucleotidetriphosphate. Preferably, the crystal has a unit cell dimension of a=83Å b=86 Å c=178 Å, α=β=γ=90° and belongs to space group P2₁2₁2₁. It willbe readily apparent to those skilled in the art that the unit cells ofthe crystal compositions may deviate ±1-2 Å from the above celldimensions depending on the deviation in the unit cell calculations.

The invention also provides a crystal composition comprisingphosphorylated GSK-3βprotein or its homologue with or without a chemicalentity. Preferably, the chemical entity is an inhibitor for the activesite, an ATP analogue or nucleotide triphosphate. Preferably, the unitcell dimensions of the crystal is a=64 Å b=67 Å c=67 Å α=100 ° β=103°γ=89.8° or a=64 Å b=67 Å c=67 Å α=80° β=77° γ=89.8° and belongs to thespace group P1. In another embodiment, the chemical entity is asubstrate or inhibitor to the substrate binding groove, or a peptidecomprising a phosphorylation sequence. Preferably, the chemical entityis HSSPHQpSEDEEE (SEQ ID NO: 21) and the unit cell dimensions of thecrystal is a=75 Åb=108 Å c=121 Å α=β=γ90° and belongs to the space groupP2₁2₁2₁. It will be readily apparent to those skilled in the art thatthe unit cells of the crystal compositions may deviate ±1-2 Å or ±1-2°from the above cell dimensions depending on the deviation in the unitcell calculations.

As used herein, the GSK-3β protein in the crystallizable or crystalcompositions can be the full length GSK-3β protein (amino acids 1-420 ofSEQ ID NO: 1); a truncated GSK-3β protein with amino acids 7-420,25-381, 37-381 of SEQ ID NO: 1; the full length protein withconservative substitutions; said truncated protein with conservativemutations. In one embodiment, the GSK-3β protein is produced from thebaculovirus system. The unphosphorylated GSK-3β protein is notphosphorylated at any of the phosphorylation sites. The phosphorylatedGSK-3β protein is phosphorylated at any of the phosphorylation sites,for example, at Serine 9 or Tyrosine 216. Preferably, the protein isphosphorylated at Tyrosine 216.

The GSK-3β protein or its homologue may be produced by any well-knownmethod, including synthetic methods, such as solid phase, liquid phaseand combination solid phase/liquid phase syntheses; recombinant DNAmethods, including cDNA cloning, optionally combined with site directedmutagenesis; and/or purification of the natural products. In oneembodiment, the protein is overexpressed from a baculovirus system or E.Coli system.

The invention also relates to a method of obtaining a crystal of aGSK-30 protein complex or GSK-3β homologue protein complex comprising achemical entity that binds to the substrate-binding groove, comprisingthe steps of:

a) producing and purifying a GSK-3β protein;

b) mixing a crystallization solution with the protein complex to producea crystallizable composition; and

c) subjecting the composition to conditions which promotecrystallization.

Conditions for promoting crystallization include, for example,apparatuses and devices for forming crystals, for example, a hangingdrop, sitting drop, dialysis or microtube batch device, will promotecrystallization. (U.S. Pat. Nos. 4,886,646, 5,096,676, 5,130,105,5,221,410 and 5,400,741; Pav et al., Proteins: Structure, Function, andGenetics, 20, pp. 98-102 (1994), incorporated herein by reference). Thehanging drop or sitting drop methods produce crystals by vapordiffusion. The hanging drop or sitting drop which contains thecrystallizable composition is equilibrated against a reservoircontaining a higher concentration of precipitant. As the drop approachesequilibrium with the reservoir, the protein becomes saturated insolution and crystals form. One of ordinary skilled in the art would beable to vary the crystallization conditions disclosed above and identifyother crystallization conditions that would produce crystals for GSK-3protein or its homologues with or without a chemical entity. Suchvariations may include adjusting the pH, salt type or concentration,precipitant type or concentration, crystallization temperature, proteinconcentration. One may also use high throughput crystallization assaysto assist in finding or optimizing the crystallization condition.

Binding Pockets of GSK-3β Protein, Protein Complexes or HomologuesThereof

As disclosed above, applicants have provided the three-dimensional X-raycrystal structures of unphosphorylated GSK-3β, phosphorylated GSK-3β,unphosphorylated GSK-3β-inhibitor complexes, phosphorylatedGSK-3β-inhibitor complex and phosphorylated GSK-3β-ADP-peptide complex.The crystal structure of GSK-3β presented here is within the GSK-3subfamily. The invention will be useful for inhibitor design. The atomiccoordinate data is presented in FIGS. 1-7.

In order to use the structure coordinates generated for theunphosphorylated and phosphorylated GSK-3β, their complexes or one ofits binding pockets or GSK-3β-like binding pocket thereof, it is oftentimes necessary to convert them into a three-dimensional shape. This isachieved through the use of commercially available software that iscapable of generating three-dimensional structures of molecules orportions thereof from a set of structure coordinates.

Binding pockets, also referred to as binding sites in the presentinvention, are of significant utility in fields such as drug discovery.The association of natural ligands or substrates with the bindingpockets of their corresponding receptors or enzymes is the basis of manybiological mechanisms of action. Similarly, many drugs exert theirbiological effects through association with the binding pockets ofreceptors and enzymes. Such associations may occur with all or part ofthe binding pocket. An understanding of such associations will help leadto the design of drugs having more favorable associations with theirtarget receptor or enzyme, and thus, improved biological effects.Therefore, this information is valuable in designing potentialinhibitors of the binding pockets of biologically important targets. Thebinding pockets of this invention will be important for drug design.

The structure coordinates described above may be used to derive thetorsion angles of the side chains (S. C. Lovell et al, Proteins:Structure, Function, and Genetics, 40, 389-408, (2000)). For example, inGlutamine, χ1 defines the torsion angle between N, Cα, Cβ, Cγ; χ2defines the torsion angle between Cα, cβ, Cγ, Cδ; and χ3 defines thetorsion angle between Cβ, Cγ, Cδ, Oε.

Surprisingly, it has now been found that for GSK-3β-inhibitor4 complex(FIG. 6), the conformation of Gln185 is very different from theconformations reported for glutamines at this position inunphosphorylated, phosphorylated GSK-3β, GSK-3β-ADP-peptide complex andother protein kinases. A glutamine side chain is able to adopt differentconformations, depending on its chemical environment. In the case ofGln185, the molecule that occupies the active site influences theconformation of the side chain of glutamine. When the molecule thatoccupies the GSK-3β active site contains an ortho-substituted phenylring and that ring is within 3.9 Å of Ile 62, Phe 67, Val 70, Asn 186and Asp 200, the glutamine side chain adopts a conformation with a χ1angle of −176.4° and a χ2 angle of 174°. Taking into consideration thesteric hindrance of nearby residues, χ1 of Gln185 can range from 123° to180°, χ2 can range from −174° to −180° and 106° to 180°. The χ1 can alsorange from −100° to −180°, and χ2 can range from −151° to −180° and 126°to 180°.

In order to compare the conformations of GSK-3β and other proteinkinases at a particular amino acid site, such as Gln185, along thepolypeptide backbone, well-known procedures may be used for doingsequence alignments of the amino acids. Such sequence alignments allowfor the equivalent sites to be compared. One such method for doing asequence alignment is the “bestfit” program available from GeneticsComputer Group which uses the local homology algorithm described bySmith and Waterman in Advances in Applied Mathematics 2; 482 (1981).

Equivalents of the Gln185 residue of GSK-3β may also be identified byits functional position. Gln185 is located on the α-helical domain ofthe GSK-3β kinase domain in front of the active site. It is positionedfive residues after the conserved RD motif (Arg 180, Asp 181) and justbefore the beginning of beta-strand β7 (Bax, B. et al. Structure, 9, pp1143-1152, (2001)).

A comparison of the torsion angles between Gln185 in the GSK-3β orGSK-3β complexes and those of corresponding glutamines in other kinasesare illustrated in Table 2. The torsion angles were determined by theprogram QUANTA.

TABLE 2 Protein χ1 (°) χ2 (°) χ3 (°) GSK-3β-peptide-ADP −60.0 83.3 −25.7Phosphorylated GSK-3β −59.9 83.2 −22.5 Unphosphorylated GSK-3β −60.963.0 88.7 GSK-3β-inhibitor1 −67.1 113.9 −72.2 GSK-3β-inhibitor2 −60.790.1 98 GSK-3β-inhibitor3 −63.3 −158.5 179.8 GSK-3β-inhibitor4 −176.4−174.0 −3.3 CDK2_inhibitor¹ 87.4 −172.5 73.4 CDK2_cyclin A² −98.1 −59.271.2 ¹Cyclin-Dependant Kinase 2 in complex with oxindole inhibitor.Davis et al., Science, 291, 134 (2001); Protein Data Bank Accessionnumber 1FVV. ²Phosphorylated Cyclin-Dependent Kinase-2 bound to CyclinA. Russo et al., Nat. Struct. Biol., 3, 696 (1996); Protein Data BankAccession number 1JST.

In the crystal structure of the phosphorylated GSK-3β-inhibitor1complex, amino acid residues I62, G63, F67, V70, A83, K85, V110, L132,D133, Y134, V135, T138, N186, L188, C199, and D200 according to FIG. 3were within 5 Å of the inhibitor bound in the active site. These aminoacids residues were identified using the program QUANTA (MolecularSimulations, Inc., San Diego, Calif. ©1998), O (Jones et al., ActaCrystallogr. A47, pp. 110-119 (1991)) and RIBBONS (Carson, J. Appl.Crystallogr., 24, pp. 9589-961 (1991)). The programs allow the displayand output of all residues within 5 Å from the inhibitor. In addition,amino acid residues V61, I62, G63, N64, G65, F67, V69, V70, A83, K85,K86, V87, E97, M101, V110, R111, L130, V131, L132, D133, Y134, V135,P136, E137, T138, R141, D181, K183, Q185, N186, L187, L188, L189, K197,L198, C199, D200, F201 and G202 according to FIG. 3 were within 8 Å ofthe inhibitor bound in the active site. These amino acid residues wereidentified using the programs QUANTA, O and RIBBONS, supra.

In the crystal structure of the unphosphorylated GSK-3β-inhibitor2complex, amino acid residues I62, G63, V70, A83, V110, L132, D133, Y134,V135, P136, E137, T138, R141, L188 and C199 according to FIG. 4 werewithin 5 Å of the inhibitor bound in the active site. These amino acidresidues were identified using the programs QUANTA, O and RIBBONS. Inaddition, amino acid residues V61, 162, G63, N64, G65, G68, V69, V70,Y71, Q72, L81, V82, A83, I84, K85, E97, M101, V110, R111, L130, V131,L132, D133, Y134, V135, P136, E137, T138, V139, Y140, R141, Q185, N186,L187, L188, L189, K197, L198, C199, D200 and F201 according to FIG. 4were within 8 Å of the inhibitor bound in the active site. These aminoacids residues were identified using the programs QUANTA, O and RIBBONS.

In the crystal structure of the unphosphorylated GSK-3β-inhibitor3complex, amino acid residues I62, N64, G65, S66, F67, G68, V69, V70,A83, K85, K86, V87, E97, V110, L132, D133, Y134, V135, P136, E137, T138,R141, K183, Q185, N186, L188, C199 and D200 according to FIG. 5 werewithin 5 Å of the inhibitor bound in the active site. These amino acidresidues were identified using the programs QUANTA, O and RIBBONS. Inaddition, amino acid residues V61, 162, G63, N64, G65, S66, F67, G68,V69, V70, Y71, Q72, L81, V82, A83, I84, K85, K86, V87, L88, E97, M101,V110, R111, L130, V131, L132, D133, Y134, V135, P136, E137, T138, V139,Y140, R141, H179, D181, K183, Q185, N186, L187, L188, L189, K197, L198,C199, D200, F201, G202, S203 and S219 according to FIG. 5 were within 8Å of the inhibitor bound in the active site. These amino acid residueswere identified using the programs QUANTA, O and RIBBON.

In the crystal structure of the unphosphorylated GSK-3β-inhibitor4complex, amino acid residues I62, G63, N64, F67, V70, A83, V110, L132,D133, Y134, V135, P136, E137, T138, R141, Q185, N186, L188, C199 andD200 according to FIG. 6 were within 5 Å of the inhibitor bound in theactive site. These amino acid residues were identified using theprograms QUANTA, O and RIBBONS. In addition, applicants have determinedthat amino acid residues V61, 162, G63, N64, G65, S66, F67, G68, V69,V70, Y71, Q72, L81, V82, A83, I84, K85, E97, M101, V110, R111, L130,V131, L132, D133, Y134, V135, P136, E137, T138, V139, Y140, R141, D181,K183, P184, Q185, N186, L187, L188, L189, K197, L198, C199, D200 andF201 according to FIG. 6 were within 8 Å of the inhibitor bound in theactive site. These amino acid residues were identified using theprograms QUANTA, O and RIBBONS.

Using a multiple alignment program to compare the unphosphorylatedGSK-3β structure and structures of other members of the protein kinasefamily, amino acid residues Y56, T59, K60, V61, V69, V70, Y71, Q72, A73,K74, L75, L81, V82, A83, 184, K85, K86, L98, M101, R102, L104, H106,C107, N108, I109, V110, R111, L112, R113, Y114, F115, F116, L128, N129,L130, V131, L132, D133, Y134, V135, P136, E137, T138, V139, Y140, R141,V142, P154, V155, I156, Y157, V158, K159, L160, Y161, M162, Y163, Q164,L165, F166, R167, S168, L169, A170, Y171, I172, H173, S174, F175, G176,I177, C178, H179, R180, D181, I182, K183, P184, Q185, N186, L187, L188,L189, D190, P191, A194, V195, L196, K197, L198, C199 and D200 accordingto FIG. 1 were identified as the ATP-binding pocket (Gerstein et al., J.Mol. Biol., 251, pp. 161-175 (1995), incorporated herein by reference).To perform this comparison, first, a sequence alignment between membersof the protein kinase family was performed. Second, a putative core wasconstructed by superimposing a series of corresponding structures in theprotein kinase family. Third, residues of high spatial variation werediscarded, and the core alignment was iteratively refined. The aminoacids that make up the final core structure have low structural varianceand have similar local and global conformation relative to thecorresponding residues in the protein family.

In the crystal structure of the phosphorylated GSK-3β-ADP-peptidecomplex, amino acids residues G65, S66, F67, D90, K91, R92, F93, K94,R96, R180, D181, K183, G202, S203, K205, P212, N213, V214, Y216, I217,C218, S219, R223, Y234 according to FIG. 7 were within 5 Å of thepeptide bound in the substrate binding groove. These amino acid residueswere identified using the programs QUANTA, O and RIBBONS, supra. Aminoacid residues D90, K91, R92, F93, K94 made backbone interactions withthe peptide substrate. Amino acid residues R96, R180, K205, N213, Y234bound to pS656 of the peptide substrate. Amino acid residues R96, R180and K205 formed a positively charged binding pocket surrounding thepS656. Amino acid residue S66 bound to the backbone of amino acidresidue S652 from the peptide substrate. Amino acid residues F67, F93form an aromatic binding pocket surrounding the peptide substrate aminoacid residue H650.

In the crystal structure of the phosphorylated GSK-3β-ADP-peptidecomplex, amino acid residues N64, G65, S66, F67, G68, V87, L88, D90,K91, R92, F93, K94, N95, R96, E97, R180, D181, I182, K183 Q185, N186,D200, F201, G202, S203, A204, K205, Q206, L207, E211, P212, N213, V214,S215, Y216, I217, C218, S219, R220, Y221, Y222, R223, L227, T232 andY234 according to FIG. 5 were within 8 Å of the peptide bound in thesubstrate binding groove. These amino acid residues were identifiedusing the programs QUANTA, O and RIBBONS, supra. Amino acid residuesY216, I217, C218, S219, R220 and R223 form a binding pocket thataccommodates the proline side chain of the peptide substrate.

In the GSK-3β-ADP-peptide electron density map, the side chains ofresidues F67, K91, and R92 in the substrate binding pocket could not belocated. Alanine and glycine residues were used to build the structuremodel at these positions. For the purpose of this invention, thestructure coordinates of amino acid residues F67, K91 and R92 refer tothe structure coordinates of amino acid residues A67, A91 and G92 inFIG. 7, respectively. In FIGS. 1-7, where alanine or glycine residueswere built in the model as a result of missing side chains in theelectron density map, the same applies to those residues.

In one embodiment, the inhibitor-binding pocket comprises GSK-3β aminoacid residues K85, M101, V110, L130 and L132 according to any one ofFIGS. 1-7. In another embodiment, the inhibitor-binding pocket comprisesGSK-3β amino acid residues I62, V135, P136, T138 and L188 according toany one of FIGS. 1-7. In another embodiment, the inhibitor-bindingpocket comprises GSK-3β amino acid residues V70, V110, L188 and C199according to any one of FIGS. 1-7. In another embodiment, theinhibitor-binding pocket comprises GSK-3β amino acids F67, V70, Q185 andC199 according to any one of FIGS. 1-7.

In one embodiment the inhibitor-binding pocket comprises amino acidresidues V61, 162, G63, N64, G65, S66, F67, G68, V69, V70, Y71, Q72,L81, V82, A83, 184, K85, K86, V87, L88, E97, M101, V110, R111, L112,L130, V131, L132, D133, Y134, V135, P136, E137, T138, V139, Y140, R141,H179, D181, K183, P184, Q185, N186, L187, L188, L189, K197, L198, C199,D200, F201, G202, S203 and S219 according to any one of FIGS. 1-7.

In another embodiment, the ATP-binding pocket comprises amino acidresidues Y56, T59, K60, V61, V69, V70, Y71, Q72, A73, K74, L75, L81,V82, A83, I84, K85, K86, L98, M101, R102, L104, H106, C107, N108, I109,V110, R111, L112, R113, Y114, F115, F116, L128, N129, L130, V131, L132,D133, Y134, V135, P136, E137, T138, V139, Y140, R141, V142, P154, V155,I156, Y157, V158, K159, L160, Y161, M162, Y163, Q164, L165, F166, R167,S168, L169, A170, Y171, I172, H173, S174, F175, G176, I177, C178, H179,R180, D181, I182, K183, P184, Q185, N186, L187, L188, L189, D190, P191,A194, V195, L196, K197, L198, C199 and D200 according to any one ofFIGS. 1-7.

In another embodiment, the substrate binding pocket comprises amino acidresidues S66, F67 and F93 according to any one of FIGS. 1-7. In anotherembodiment, the substrate binding pocket comprises amino acid residuesG65, S66, F67 and F93 according to any one of FIGS. 1-7.

In another embodiment, the substrate binding pocket comprises amino acidresidues D90, K91, R92, F93, K94 according to any one of FIGS. 1-7.

In another embodiment, the substrate binding pocket comprises amino acidresidues R96, R180, K205, N213 and Y234 according to any one of FIGS.1-7. In another embodiment, the substrate binding pocket comprises aminoacid residues R96, R180 and K205 according to any one of FIGS. 1-7.

In another embodiment, the substrate binding pocket comprises amino acidresidues Y216, I217, C218, S219, R220 and R223 according to any one ofFIGS. 1-7.

In another embodiment, the substrate binding pocket comprises amino acidresidues G65, S66, F67, D90, K91, R92, F93, K94, R96, R180, D181, K183,G202, S203, K205, P212, N213, V214, Y216, I217, C218, S219, R223 andY234 according to any one of FIGS. 1-7.

In yet another embodiment, the substrate binding pocket comprises aminoacid residues N64, G65, S66, F67, G68, V87, L88, D90, K91, R92, F93,K94, N95, R96, E97, R180, D181, I182, K183 Q185, N186, D200, F201, G202,S203, A204, K205, Q206, L207, E211, P212, N213, V214, S215, Y216, I217,C218, S219, R220, Y221, Y222, R223, L227, T232 and Y234 according to anyone of FIGS. 1-7.

Thus, the binding pockets of this invention are defined by the structurecoordinates of the above amino acids, as set forth in FIGS. 1-7.

It will be readily apparent to those of skill in the art that thenumbering of amino acid residues in other homologues of GSK-3β may bedifferent than that set forth for GSK-3β. Corresponding amino acidresidues in homologues of GSK-3β are easily identified by visualinspection of the amino acid sequences or by using commerciallyavailable homology software programs.

Those of skill in the art understand that a set of structure coordinatesfor an enzyme or an enzyme-complex or a portion thereof, is a relativeset of points that define a shape in three dimensions. Thus, it ispossible that an entirely different set of coordinates could define asimilar or identical shape. Moreover, slight variations in theindividual coordinates will have little effect on overall shape. Interms of binding pockets, these variations would not be expected tosignificantly alter the nature of ligands that could associate withthose pockets.

The variations in coordinates discussed above may be generated becauseof mathematical manipulations of the GSK-3β structure coordinates. Forexample, the structure coordinates set forth in any one of FIGS. 1-7could be manipulated by crystallographic permutations of the structurecoordinates, fractionalization of the structure coordinates, integeradditions or subtractions to sets of the structure coordinates,inversion of the structure coordinates or any combination of the above.

Alternatively, modifications in the crystal structure due to mutations,additions, substitutions, and/or deletions of amino acids, or otherchanges in any of the components that make up the crystal could alsoaccount for variations in structure coordinates. If such variations arewithin a certain root mean square deviation as compared to the originalcoordinates, the resulting three-dimensional shape is consideredencompassed by this invention. Thus, for example, a ligand that bound tothe binding pocket of GSK-3β would also be expected to bind to anotherbinding pocket whose structure coordinates defined a shape that fellwithin the acceptable root mean square deviation.

Various computational analyses may be necessary to determine whether amolecule or the binding pocket or portion thereof is sufficientlysimilar to the GSK-3β binding pockets described above. Such analyses maybe carried out using well known software applications, such as theMolecular Similarity application of QUANTA (Molecular Simulations Inc.,San Diego, Calif. © 1998), CCP4 (Acta Crystallogr., D50, 760-763 (1994))or ProFit (A. C. R. Martin, ProFit version 1.8).

The Molecular Similarity software application permits comparisonsbetween different structures, different conformations of the samestructure, and different parts of the same structure. The procedure usedin Molecular Similarity to compare structures is divided into foursteps: 1) load the structures to be compared; 2) define the atomequivalences in these structures; 3) perform a fitting operation; and 4)analyze the results.

Each structure in the comparison is identified by a name. One structureis identified as the target (i.e., the fixed structure); all remainingstructures are working structures (i.e., moving structures). Since atomequivalency within QUANTA is defined by user input, for the purpose ofthis invention we will define equivalent atoms as protein backbone atomsN, C, O and Cα for all corresponding amino acids between the twostructures being compared.

The corresponding amino acids may be identified by sequence alignmentprograms such as the “bestfit” program available from the GeneticsComputer Group which uses the local homology algorithm described bySmith and Waterman in Advances in Applied Mathematics 2, 482 (1981),which is incorporated herein by reference. The identification ofequivalent residues can also be assisted by secondary structurealignment, for example, aligning the α-helices, β-sheets or hinge region(residues 126-135 in GSK-3β) in the structure. For programs thatcalculate RMSD values of the backbone atoms, an RMSD cutoff value can beused to exclude pairs of equivalent atoms with extreme individual RMSDvalues, or in situations where the equivalent atom can not be found inthe corresponding structure.

When a rigid fitting method is used, the working structure is translatedand rotated to obtain an optimum fit with the target structure. Thefitting operation uses an algorithm that computes the optimumtranslation and rotation to be applied to the moving structure, suchthat the root mean square difference of the fit over the specified pairsof equivalent atom is an absolute minimum. This number, given inangstroms, is reported by QUANTA.

The RMSD values of the inhibitor and substrate-binding pockets betweenthe GSK-3β-ADP-peptide structure (FIG. 7) and other GSK-3β structures(FIGS. 1-6) are illustrated in Tables 3-9. The RMSD values werecalculated by the program LSQKAB in CCP4, supra. Backbone atoms (C, O, Nand Cα) of all residues in the binding pocket were used in thecalculation of the RMSD.

TABLE 3 Inhibitor-binding pocket (K85, M101, V110, L130 and L132) RMSDbetween inhibitor GSK-3β structures binding pockets (Å) unphosphorylatedGSK-3β 0.32 phosphorylated GSK-3β 0.34 GSK-3β inhibitor1 complex 0.32GSK-3β inhibitor2 complex 0.39 GSK-3β inhibitor3 complex 0.27 GSK-3βinhibitor4 complex 0.27

TABLE 4 Inhibitor-binding pocket (I62, V135, P136, T138 and L188) RMSDbetween inhibitor GSK-3β structures binding pockets (Å) unphosphorylatedGSK-3β 1.16 phosphorylated GSK-3β 1.02 GSK-3β inhibitor1 complex 0.77GSK-3β inhibitor2 complex 0.95 GSK-3β inhibitor3 complex 0.23 GSK-3βinhibitor4 complex 0.31

TABLE 5 Inhibitor-binding pocket (F67, V70, Q185, C199) RMSD betweeninhibitor GSK-3β structures binding pockets (Å) unphosphorylated GSK-3β1.83 phosphorylated GSK-3β 1.89 GSK-3β inhibitor1 complex 1.67 GSK-3βinhibitor2 complex 2.23 GSK-3β inhibitor3 complex 1.71 GSK-3β inhibitor4complex 0.80

TABLE 6 Inhibitor-binding pocket (V70, V110, L188 and C199) RMSD betweeninhibitor GSK-3β structures binding pockets (Å) unphosphorylated GSK-3β0.80 phosphorylated GSK-3β 0.88 GSK-3β inhibitor1 complex 0.81 GSK-3βinhibitor2 complex 0.92 GSK-3β inhibitor3 complex 0.38 GSK-3β inhibitor4complex 0.30

TABLE 7 Substrate-binding pocket (G65, S66, F67 and F93) RMSD betweeninhibitor GSK-3β structures binding pockets (Å) unphosphorylated GSK-3β1.93 phosphorylated GSK-3β 1.32 GSK-3β inhibitor1 complex 1.32 GSK-3βinhibitor2 complex 1.74 GSK-3β inhibitor3 complex 1.43 GSK-3β inhibitor4complex 2.09

TABLE 8 Substrate-binding pocket (R96, R180, K205, N213 and Y234) RMSDbetween inhibitor GSK-3β structures binding pockets (Å) unphosphorylatedGSK-3β 0.67 phosphorylated GSK-3β 0.59 GSK-3β inhibitor1 complex 0.64GSK-3β inhibitor2 complex 0.71 GSK-3β inhibitor3 complex 0.71 GSK-3βinhibitor4 complex 0.65

TABLE 9 Substrate-binding pocket (Y216, I217, C218, S219, R220 and R223)RMSD between inhibitor GSK-3β structures binding pockets (Å)unphosphorylated GSK-3β 1.07 phosphorylated GSK-3β 0.36 GSK-3βinhibitor1 complex 0.46 GSK-3β inhibitor2 complex 1.35 GSK-3β inhibitor3complex 1.39 GSK-3β inhibitor4 complex 1.21

The RMSD values of the overall structure between the GSK-3β-inhibitor2structure (FIG. 4) and other GSK-3β structures (FIGS. 1-3, and 5-7) areillustrated in Table 10. The RMSD values were calculated by the programLSQKAB in CCP4, supra. Backbone atoms (C, O, N and Cα) of all residuesin the overall structure according to FIGS. 1-7 were used in thecalculation of the RMSD.

TABLE 10 RMSD of overall GSK-3β structures structure(Å) unphosphorylatedGSK-3β 0.52 phosphorylated GSK-3β 1.27 GSK-3β-ADP-peptide complex 1.94GSK-3β inhibitor1 complex 0.79 GSK-3β inhibitor3 complex 1.77 GSK-3βinhibitor4 complex 0.90

For the purpose of this invention, any molecule or molecular complex orbinding pocket thereof that is within a root mean square deviation forbackbone atoms (C, O, N and Cα) when superimposed on the relevantbackbone atoms described by structure coordinates listed in any one ofFIGS. 1-7 are encompassed by this invention.

In one embodiment, the present invention provides a molecule ormolecular complex comprising a binding pocket defined by structurecoordinates of amino acid residues which are identical to GSK-3β aminoacid residues K85, M101, V110, L130 and L132 according to any one ofFIGS. 1-7, wherein the root mean square deviation of the backbone atomsbetween said amino acid residues and said GSK-3β amino acid residues isnot greater than about 3.0 Å. In one embodiment, the RMSD is not greaterthan about 1.0 Å. In a preferred embodiment, the RMSD is not greaterthan about 0.5 Å.

In a more preferred embodiment, the root mean square deviation of thebackbone atoms between said amino acid residues and said GSK-3β aminoacid residues according to FIG. 7 is not greater than about 0.5 Å. Inone embodiment, the RMSD is not greater than about 0.2 Å.

In another embodiment, the root mean square deviation of the backboneatoms between said amino acid residues and said GSK-3β amino acidresidues according to any one of FIGS. 1-7 is not greater than about 0.3Å, and wherein at least one of said amino acid residues is not identicalto the GSK-3β amino acid residue to which it corresponds. In oneembodiment, the RMSD is not greater than about 0.2 Å.

In another embodiment, the present invention provides a molecule ormolecular complex comprising a binding pocket defined by structurecoordinates of amino acid residues which are identical to GSK-3β aminoacid residues I62, V135, P136, T138 and L188 according to any one ofFIGS. 1-7, wherein the root mean square deviation of the backbone atomsbetween said amino acid residues and said GSK-3β amino acid residues isnot greater than about 3.0 Å. In one embodiment, the RMSD is not greaterthan about 1.5 Å. In a preferred embodiment the RMSD is not greater thanabout 1.0 Å.

In a more preferred embodiment, the root mean square deviation of thebackbone atoms between said amino acid residues and said GSK-3β aminoacid residues according to FIG. 7 is not greater than about 0.8 Å. Inone embodiment, the RMSD is not greater than about 0.5 Å. In oneembodiment the RMSD is not greater than about 0.3 Å.

In another embodiment, the root mean square deviation of the backboneatoms between said amino acid residues and said GSK-3β amino acidresidues according to any one of FIGS. 1-7 is not greater than about 0.2Å, and wherein at least one of said amino acid residues is not identicalto the GSK-3β amino acid residue to which it corresponds.

In another embodiment, the present invention provides a molecule ormolecular complex comprising a binding pocket defined by structurecoordinates of amino acid residues which are identical to GSK-3β aminoacid residues V70, V110, L188 and C199 according to any one of FIGS.1-7, wherein the root mean square deviation of the backbone atomsbetween said amino acid residues and said GSK-3β amino acid residues isnot greater than about 3.0 Å. In one embodiment the RMSD is not greaterthan about 1.5 Å. In a preferred embodiment the RMSD is not greater thanabout 1.0 Å.

In a more preferred embodiment, the root mean square deviation of thebackbone atoms between said amino acid residues and said GSK-3β aminoacid residues according to FIG. 7 is not greater than about 0.6 Å. Inone embodiment, the RMSD is not greater than about 0.4 Å. In oneembodiment, the RMSD is not greater than about 0.2 Å.

In another embodiment, the root mean square deviation of the backboneatoms between said amino acid residues and said GSK-3β amino acidresidues according to any one of FIGS. 1-7 is not greater than about 0.2Å, and wherein at least one of said amino acids is not identical to theGSK-3β amino acid to which it corresponds.

In another embodiment, the present invention provides a molecule ormolecular complex comprising a binding pocket defined by structurecoordinates of amino acid residues which are identical to GSK-3β aminoacid residues V70, F67, Q185 and C199 according to any one of FIGS. 1-7,wherein the root mean square deviation of the backbone atoms betweensaid amino acid residues and said GSK-3β amino acid residues is notgreater than about 3.0 Å. In a preferred embodiment, the RMSD is notgreater than about 2.5 Å.

In a more preferred embodiment, the root mean square deviation of thebackbone atoms between said amino acid residues and said GSK-3β aminoacid residues according to FIG. 7 is not greater than about 1.6 Å. Inone embodiment, the RMSD is not greater than about 1.1 Å. In oneembodiment, the RMSD is not greater than about 0.6 Å. In one embodiment,the RMSD is not greater than about 0.2 Å.

In another embodiment, the root mean square deviation of the backboneatoms between said amino acid residues and said GSK-3β amino acidresidue according to any one of FIGS. 1-7 is not greater than about 0.3Å, and wherein at least one of said amino acid residues is not identicalto the GSK-3β amino acid residues to which it corresponds. In oneembodiment, the RMSD is not greater than about 0.2 Å.

In another embodiment, the present invention provides a molecule ormolecular complex comprising part of a binding pocket, said bindingpocket defined by structure coordinates of amino acid residues whichcorrespond to GSK-3β amino acid residues Y56, T59, K60, V61, I62, G63,N64, G65, S66, F67, G68, V69, V70, Y71, Q72, A73, K74, L75, L81, V82,A83, I84, K85, K86, V87, L88, E97, L98, M101, R102, L104, H106, C107,N108, I109, V110, R111, L112, R113, Y114, F115, F116, L128, N129, L130,V131, L132, D133, Y134, V135, P136, E137, T138, V139, Y140, R141, V142,R144, P154, V155, I156, Y157, V158, K159, L160, Y161, M162, Y163, Q164,L165, F166, R167, S168, L169, A170, Y171, I172, H173, S174, F175, G176,I177, C178, H179, R180, D181, I182, K183, P184, Q185, N186, L187, L188,L189, D190, P191, A194, V195, L196, K197, L198, C199, D200, F201, G202,S203 and S219 according to any one of FIGS. 1-7, wherein the root meansquare deviation of the backbone atoms between said amino acid residuesand said GSK-3β amino acid residues is not greater than about 0.2 Å. Ina preferred embodiment, said amino acid residues are identical to saidGSK-3β amino acid residues.

In yet another embodiment, the present invention provides a molecule ormolecular complex comprising a binding pocket defined by structurecoordinates of amino acid residues which are identical to GSK-3β aminoacid residues G65, S66, F67 and F93 according to any one of FIGS. 1-7,wherein the root mean square deviation of the backbone atoms betweensaid amino acid residues and said GSK-3β amino acid residues is notgreater than about 3.0 Å. In one embodiment, the RMSD is not greaterthan about 2.5 Å. In a preferred embodiment, the RMSD is not greaterthan about 2.0 Å.

In a more preferred embodiment, the root mean square deviation of thebackbone atoms between said amino acid residues and said GSK-3β aminoacid residues according to FIG. 7 is not greater than about 1.5 Å. Inone embodiment, the RMSD is not greater than about 1.1 Å. In oneembodiment, the RMSD is not greater than about 0.7 Å. In one embodiment,the RMSD is not greater than about 0.5 Å.

In another embodiment, the root mean square deviation of the backboneatoms between said amino acid residues and said GSK-3β amino acidresidues according to any one of FIGS. 1-7 is not greater than about 1.1Å, and wherein at least one of said amino acid residues is not identicalto the GSK-3β amino acid residues to which it corresponds. In oneembodiment, the RMSD is not greater than about 0.8 Å. In one embodiment,the RMSD is not greater than about 0.4 Å. In one embodiment, the RMSD isnot greater than about 0.2 Å.

In another embodiment, the present invention provides a molecule ormolecular complex comprising a binding pocket defined by structurecoordinates of amino acid residues which correspond to GSK-3β aminoacids R96, R180, K205, N213 and Y234 according to any one of FIGS. 1-7,wherein the root mean square deviation of the backbone atoms betweensaid amino acid residues and said GSK-3β amino acid residues is notgreater than about 3.0 Å, wherein said binding pocket comprises an aminoacid residue asparagine corresponding to said GSK-3β amino acid residueN213. In another embodiment, said amino acid residues are identical tosaid GSK-3β amino acids. In one embodiment, the RMSD is not greater thanabout 1.5 Å. In a preferred embodiment, the RMSD is not greater thanabout 1.0 Å.

In a more preferred embodiment, the root mean square deviation ofbackbone atoms between said amino acid residues and said GSK-3β aminoacid residues according to FIG. 7 is not greater than about 0.4 Å. Inone embodiment, the RMSD is not greater than about 0.2 Å.

In another embodiment, the root mean square deviation of the backboneatoms between said amino acid residues and said GSK-3β amino acidresidues according to any one of FIGS. 1-7 is not greater than about 3.0Å, and wherein at least one of said amino acid residues is not identicalto the GSK-3β amino acid residues to which it corresponds. In oneembodiment, the RMSD is not greater than about 1.2 Å. In one embodiment,the RMSD is not greater than about 0.7 Å. In one embodiment, the RMSD isnot greater than about 0.3 Å.

In another embodiment, the present invention provides a molecule ormolecular complex comprising a binding pocket defined by structurecoordinates of amino acid residues which correspond to GSK-3β amino acidresidues Y216, I217, C218, S219, R220 and R223 according to any one ofFIGS. 1-7, wherein the root mean square deviation of the backbone atomsbetween said amino acid residues and said GSK-3β amino acid residues isnot greater than about 3.0 Å, wherein said binding pocket comprises acysteine amino acid residue corresponding to said GSK-3β amino acidresidue C218. In another embodiment, said amino acid residues areidentical to said GSK-3β amino acids. In one embodiment, the RMSD is notgreater than about 2.0 Å. In a preferred embodiment, the RMSD is notgreater than about 1.5 Å.

In a more preferred embodiment, the root mean square deviation ofbackbone atoms between said amino acid residues and said GSK-3β aminoacid residues according to FIG. 5 is not greater than about 1.1 Å. Inone embodiment, the RMSD is not greater than about 0.5 Å. In oneembodiment, the RMSD is not greater than about 0.2 Å.

In another embodiment, the root mean square deviation of the backboneatoms between said amino acid residues and said GSK-3β amino acidresidues according to any one of FIGS. 1-7 is not greater than about 1.1Å, and wherein at least one of said amino acid residues is not identicalto the GSK-3β amino acid to which it corresponds. In one embodiment, theRMSD is not greater than about 0.8 Å. In one embodiment, the RMSD is notgreater than about 0.5 Å. In one embodiment, the RMSD is not greaterthan about 0.2 Å.

In another embodiment, the present invention provides a molecule ormolecular complex comprising part of a binding pocket, said bindingpocket defined by structure coordinates of amino acid residues whichcorrespond to GSK-3β amino acid residues N64, G65, S66, F67, G68, V87,L88, D90, K91, R92, F93, K94, N95, R96, E97, R180, D181, I182, K183Q185, N186, D200, F201, G202, S203, A204, K205, Q206, L207, E211, P212,N213, V214, S215, Y216, I217, C218, S219, R220, Y221, Y222, R223, L227,T232 and Y234 according to any one of FIGS. 1-7, wherein the root meansquare deviation of the backbone atoms between said amino acid residuesand said GSK-3β amino acid residues is not greater than about 1.0 Å. Inone embodiment, the RMSD is not greater than about 0.7 Å. In oneembodiment, the RMSD is not greater than about 0.5 Å. In one embodiment,the RMSD is not greater than about 0.2 Å. In one embodiment, said aminoacid residues are identical to said GSK-3β amino acid residues.

In one embodiment, the present invention provides a molecule ormolecular complex comprising a GSK-3β protein defined by structurecoordinates of the amino acid residues which correspond to GSK-3β aminoacid residues according to any one of FIGS. 1-7, wherein the root meansquare deviation of the backbone atoms between said amino acid residuesand said GSK-3β amino acid residues is not greater than about 2.0 Å. Inone embodiment, the RMSD is not greater than about 1.7 Å. In oneembodiment, the RMSD is not greater than about 1.1 Å. In one embodiment,the RMSD is not greater than about 0.7 Å. In one embodiment, said aminoacid residues are identical to said GSK-3β amino acid residues.

In one embodiment, the present invention provides a molecule ormolecular complex comprising a protein kinase comprising a glutamine orglutamic acid residue that corresponds to Gln185 of GSK-3β protein,wherein the χ1 angle is in the range of 123° to 180°, and the χ2 angleis in the range of −174° to −180° and 106° to 180°. In anotherembodiment, the χ1 angle is in the range of −100° to −180° and the χ2angle is in the range of −151° to −180° and 126° to 180°.

In one embodiment, the above molecules or molecular complexes are GSK-3βproteins or a GSK-3β homologues. In another embodiment, the abovemolecules or molecular complexes are in crystalline form.

Computer Systems

According to another embodiment, this invention provides amachine-readable data storage medium, comprising a data storage materialencoded with machine-readable data, wherein said data defines the abovementioned molecules or molecular complexes. In one embodiment, the datadefines the above mentioned binding pockets by comprising the structurecoordinates of said amino acid residues according to any one of FIGS.1-7.

To use the structure coordinates generated for the GSK-3β, homologuesthereof, or one of its binding pockets, it is sometimes necessary toconvert them into a three-dimensional shape. This is achieved throughthe use of commercially available software that is capable of generatinga three-dimensional structure of molecules or portions thereof from aset of structure coordinates. The three-dimensional structure may bedisplayed as a graphical representation.

Therefore, according to another embodiment, this invention provides amachine-readable data storage medium comprising a data storage materialencoded with machine readable data. In one embodiment, a machineprogrammed with instructions for using said data, is capable ofgenerating a three-dimensional structure of any of the molecule ormolecular complexes, or binding pockets thereof, that are describedherein.

This invention also provides a computer comprising:

-   -   a) a machine-readable data storage medium comprising a data        storage material encoded with machine-readable data, wherein        said data defines any one of the above binding pockets of the        molecule or molecular complex;    -   b) a working memory for storing instructions for processing said        machine-readable data;    -   c) a central processing unit coupled to said working memory and        to said machine-readable data storage medium for processing said        machine readable data; and    -   d) output hardware coupled to said central processing unit for        outputting information of said binding pocket or information        produced by using said binding pocket.

Information of said binding pocket or information produced by using saidbinding pocket can be outputted through a display terminal, printer ordisk drive. The information can be in graphical or alphanumeric form. Inanother embodiment, the computer further comprises a commerciallyavailable software program to display the information as a graphicalrepresentation. Examples of software programs include but are notlimited to QUANTA (Molecular Simulations, Inc., San Diego, Calif.©2001), O (Jones et al., Acta Crystallogr. A47, pp. 110-119 (1991)) andRIBBONS (Carson, J. Appl. Crystallogr., 24, pp. 9589-961 (1991)), whichare incorporated herein by reference.

FIG. 22 demonstrates one version of these embodiments. System (10)includes a computer (11) comprising a central processing unit (“CPU”)(20), a working memory (22) which may be, e.g., RAM (random-accessmemory) or “core” memory, mass storage memory (24) (such as one or moredisk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”)display terminals (26), one or more keyboards (28), one or more inputlines (30), and one or more output lines (40), all of which are,interconnected by a conventional bi-directional system bus (50).

Input hardware (35), coupled to computer (11) by input lines (30), maybe implemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems (32)connected by a telephone line or dedicated data line (34). Alternativelyor additionally, the input hardware (35) may comprise CD-ROM drives ordisk drives (24). In conjunction with display terminal (26), keyboard(28) may also be used as an input device.

Output hardware (46), coupled to computer (11) by output lines (40), maysimilarly be implemented by conventional devices. By way of example,output hardware (46) may include CRT display terminal (26) fordisplaying a graphical representation of a binding pocket of thisinvention using a program such as QUANTA as described herein. Outputhardware might also include a printer (42), so that hard copy output maybe produced, or a disk drive (24), to store system output for later use.

In operation, CPU (20) coordinates the use of the various input andoutput devices (35), (46), coordinates data accesses from mass storage(24) and accesses to and from working memory (22), and determines thesequence of data processing steps. A number of programs may be used toprocess the machine-readable data of this invention. Such programs arediscussed in reference to the computational methods of drug discovery asdescribed herein. Specific references to components of the hardwaresystem (10) are included as appropriate throughout the followingdescription of the data storage medium.

FIG. 23 shows a cross section of a magnetic data storage medium (100)which can be encoded with a machine-readable data that can be carriedout by a system such as system (10) of FIG. 22. Medium (100) can be aconventional floppy diskette or hard disk, having a suitable substrate(101), which may be conventional, and a suitable coating (102), whichmay be conventional, on one or both sides, containing magnetic domains(not visible) whose polarity or orientation can be altered magnetically.Medium (100) may also have an opening (not shown) for receiving thespindle of a disk drive or other data storage device (24).

The magnetic domains of coating (102) of medium (100) are polarized ororiented so as to encode in manner which may be conventional, machinereadable data such as that described herein, for execution by a systemsuch as system (10) of FIG. 22.

FIG. 24 shows a cross section of an optically-readable data storagemedium (110) which also can be encoded with such a machine-readabledata, or set of instructions, which can be carried out by a system suchas system (10) of FIG. 22. Medium (110) can be a conventional compactdisk read only memory (CD-ROM) or a rewritable medium such as amagneto-optical disk which is optically readable and magneto-opticallywritable. Medium (100) preferably has a suitable substrate (111), whichmay be conventional, and a suitable coating (112), which may beconventional, usually of one side of substrate (111).

In the case of CD-ROM, as is well known, coating (112) is reflective andis impressed with a plurality of pits (113) to encode themachine-readable data. The arrangement of pits is read by reflectinglaser light off the surface of coating (112). A protective coating(114), which preferably is substantially transparent, is provided on topof coating (112).

In the case of a magneto-optical disk, as is well known, coating (112)has no pits (113), but has a plurality of magnetic domains whosepolarity or orientation can be changed magnetically when heated above acertain temperature, as by a laser (not shown). The orientation of thedomains can be read by measuring the polarization of laser lightreflected from coating (112). The arrangement of the domains encodes thedata as described above.

Thus, in accordance with the present invention, data capable ofgenerating the three dimensional structure of the above molecules ormolecular complexes, or binding pockets thereof, can be stored in amachine-readable storage medium, which is capable of displaying agraphical three-dimensional representation of the structure.

Rational Drug Design

The GSK-3β X-ray coordinate data, when used in conjunction with acomputer programmed with software to generate those coordinates into thethree-dimensional structure of GSK-3β may be used for a variety ofpurposes, such as drug discovery. The coordinate data themselves mayalso be used directly to perform computer modelling and fittingoperations.

For example, the structure encoded by the data may be computationallyevaluated for its ability to associate with chemical entities. Chemicalentities that associate with GSK-3β may inhibit GSK-3β and itshomologues, and are potential drug candidates. Alternatively, thestructure encoded by the data may be displayed in a graphicalthree-dimensional representation on a computer screen. This allowsvisual inspection of the structure, as well as visual inspection of thestructure's association with chemical entities.

Thus, according to another embodiment, the invention relates to a methodfor evaluating the potential of a chemical entity to associate with amolecule or molecular complex as described previously in the differentembodiments.

This method comprises the steps of: a) employing computational means toperform a fitting operation between the chemical entity and a bindingpocket of the molecule or molecular complex; b) analyzing the results ofsaid fitting operation to quantify the association between the chemicalentity and the binding pocket; and optionally c) outputting saidquantified association to a suitable output hardware, such as a CRTdisplay terminal, a printer or a disk drive, as described previously.The method may further comprise the step of generating athree-dimensional graphical representation of the molecule or molecularcomplex or binding pocket thereof prior to step a).

Alternatively, the structure coordinates of the above binding pocketscan be utilized in a method for identifying an agonist or antagonist ofa molecule comprising any of the above binding pockets. This methodcomprises the steps of:

-   -   a) using the three-dimensional structure of said molecule or        molecular complex to design or select a chemical entity;    -   b) contacting said chemical entity with the molecule or        molecular complex and monitoring the activity of the molecule or        molecular complex; and    -   c) classifying said chemical entity as an agonist or antagonist        based on the effect of said chemical entity on the activity of        the molecule or molecular complex.

In one embodiment, step a) is using the three-dimensional structure ofthe binding pocket of the molecule or molecular complex. In anotherembodiment, the three-dimensional structure is displayed as a graphicalrepresentation.

The present invention permits the use of molecular design techniques toidentify, select and design chemical entities, including inhibitorycompounds, capable of binding to the above binding pockets.

The elucidation of binding pockets on GSK-3β provides the necessaryinformation for designing new chemical entities and compounds that mayinteract with GSK-3β substrate or ATP-binding pockets, in whole or inpart. Due to the homology in the kinase core of GSK-3β and GSK-3α,compounds that inhibit GSK-3β are also expected to inhibit GSK-3α,especially those compounds that bind the ATP-binding pocket.

Throughout this section, discussions about the ability of an entity tobind to, associate with or inhibit the above binding pockets refers tofeatures of the entity alone. Assays to determine if a compound binds toGSK-3β are well known in the art and are exemplified below.

The design of compounds that bind to or inhibit the above bindingpockets according to this invention generally involves consideration oftwo factors. First, the entity must be capable of physically andstructurally associating with parts or all of the above binding pockets.Non-covalent molecular interactions important in this associationinclude hydrogen bonding, van der Waals interactions, hydrophobicinteractions and electrostatic interactions.

Second, the entity must be able to assume a conformation that allows itto associate with the above binding pockets directly. Although certainportions of the entity will not directly participate in theseassociations, those portions of the entity may still influence theoverall conformation of the molecule. This, in turn, may have asignificant impact on potency. Such conformational requirements includethe overall three-dimensional structure and orientation of the chemicalentity in relation to all or a portion of the binding pocket, or thespacing between functional groups of an entity comprising severalchemical entities that directly interact with the above binding pockets.

The potential inhibitory or binding effect of a chemical entity on theabove binding pockets may be analyzed prior to its actual synthesis andtesting by the use of computer modeling techniques. If the theoreticalstructure of the given entity suggests insufficient interaction andassociation between it and the above binding pockets, testing of theentity is obviated. However, if computer modeling indicates a stronginteraction, the molecule may then be synthesized and tested for itsability to bind to the above binding pocket. This may be achieved bytesting the ability of the molecule to inhibit GSK-3β using the assaysdescribed in Example 18. In this manner, synthesis of inoperativecompounds may be avoided.

A potential inhibitor of the above binding pockets may becomputationally evaluated by means of a series of steps in whichchemical entities or fragments are screened and selected for theirability to associate with the above binding pockets.

One skilled in the art may use one of several methods to screen chemicalentities or fragments for their ability to associate with the abovebinding pockets. This process may begin by visual inspection of, forexample, any of the above binding pockets on the computer screen basedon the GSK-3β structure coordinates in any one of FIGS. 1-7 or othercoordinates which define a similar shape generated from themachine-readable storage medium. Selected fragments or chemical entitiesmay then be positioned in a variety of orientations, or docked, withinthat binding pocket as defined supra. Docking may be accomplished usingsoftware such as QUANTA and Sybyl, followed by energy minimization andmolecular dynamics with standard molecular mechanics force fields, suchas CHARMM and AMBER.

Specialized computer programs may also assist in the process ofselecting fragments or chemical entities. These include:

-   -   1. GRID (P. J. Goodford, “A Computational Procedure for        Determining Energetically Favorable Binding Sites on        Biologically Important Macromolecules”, J. Med. Chem., 28, pp.        849-857 (1985)). GRID is available from Oxford University,        Oxford, UK.    -   2. MCSS (A. Miranker et al., “Functionality Maps of Binding        Sites: A Multiple Copy Simultaneous Search Method.” Proteins:        Structure, Function and Genetics, 11, pp. 29-34 (1991)). MCSS is        available from Molecular Simulations, San Diego, Calif.    -   3. AUTODOCK (D. S. Goodsell et al., “Automated Docking of        Substrates to Proteins by Simulated Annealing”, Proteins:        Structure, Function, and Genetics, 8, pp. 195-202 (1990)).        AUTODOCK is available from Scripps Research Institute, La Jolla,        Calif.    -   4. DOCK (I. D. Kuntz et al., “A Geometric Approach to        Macromolecule-Ligand Interactions”, J. Mol. Biol., 161, pp.        269-288 (1982)). DOCK is available from University of        California, San Francisco, Calif.

Once suitable chemical entities or fragments have been selected, theycan be assembled into a single compound or complex. Assembly may bepreceded by visual inspection of the relationship of the fragments toeach other on the three-dimensional image displayed on a computer screenin relation to the structure coordinates of GSK-3β. This would befollowed by manual model building using software such as QUANTA or Sybyl(Tripos Associates, St. Louis, Mo.).

Useful programs to aid one of skill in the art in connecting theindividual chemical entities or fragments include:

-   -   1. CAVEAT (P. A. Bartlett et al., “CAVEAT: A Program to        Facilitate the Structure-Derived Design of Biologically Active        Molecules”, in “Molecular Recognition in Chemical and Biological        Problems”, Special Pub., Royal Chem. Soc., 78, pp. 182-196        (1989); G. Lauri and P. A. Bartlett, “CAVEAT: a Program to        Facilitate the Design of Organic Molecules”, J. Comput. Aided        Mol. Des., 8, pp. 51-66 (1994)). CAVEAT is available from the        University of California, Berkeley, Calif.    -   2. 3D Database systems such as ISIS (MDL Information Systems,        San Leandro, Calif.). This area is reviewed in Y. C. Martin, “3D        Database Searching in Drug Design”, J. Med. Chem., 35, pp.        2145-2154 (1992).    -   3. HOOK (M. B. Eisen et al., “HOOK: A Program for Finding Novel        Molecular Architectures that Satisfy the Chemical and Steric        Requirements of a Macromolecule Binding Site”, Proteins:        Struct., Funct., Genet., 19, pp. 199-221 (1994). HOOK is        available from Molecular Simulations, San Diego, Calif.

Instead of proceeding to build an inhibitor of any of the above bindingpockets in a step-wise fashion, one fragment or chemical entity at atime as described above, inhibitory or other GSK-3β binding compoundsmay be designed as a whole or “de novo” using either an empty bindingpocket or optionally including some portion(s) of a known inhibitor(s).There are many de novo ligand design methods including:

-   -   1. LUDI (H.-J. Bohm, “The Computer Program LUDI: A New Method        for the De Novo Design of Enzyme Inhibitors”, J. Comp. Aid.        Molec. Design, 6, pp. 61-78 (1992)). LUDI is available from        Molecular Simulations Incorporated, San Diego, Calif.    -   2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985        (1991)). LEGEND is available from Molecular Simulations        Incorporated, San Diego, Calif.    -   3. LeapFrog (available from Tripos Associates, St. Louis, Mo.).    -   4. SPROUT (V. Gillet et al., “SPROUT: A Program for Structure        Generation)”, J. Comput. Aided Mol. Design, 7, pp. 127-153        (1993)). SPROUT is available from the University of Leeds, UK.

Other molecular modeling techniques may also be employed in accordancewith this invention (see, e.g., N. C. Cohen et al., “Molecular ModelingSoftware and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp.883-894 (1990); see also, M. A. Navia and M. A. Murcko, “The Use ofStructural Information in Drug Design”, Current Opinions in StructuralBiology, 2, pp. 202-210 (1992); L. M. Balbes et al., “A Perspective ofModern Methods in Computer-Aided Drug Design”, in Reviews inComputational Chemistry, Vol. 5, K. B. Lipkowitz and D. B. Boyd, Eds.,VCH, New York, pp. 337-380 (1994); see also, W. C. Guida, “Software ForStructure-Based Drug Design”, Curr. Opin. Struct. Biology, 4, pp.777-781 (1994)).

Once a compound has been designed or selected by the above methods, theefficiency with which that entity may bind to any of the above bindingpockets may be tested and optimized by computational evaluation. Forexample, an effective binding pocket inhibitor must preferablydemonstrate a relatively small difference in energy between its boundand free states (i.e., a small deformation energy of binding). Thus, themost efficient binding pocket inhibitors should preferably be designedwith a deformation energy of binding of not greater than about 10kcal/mole, more preferably, not greater than 7 kcal/mole. Binding pocketinhibitors may interact with the binding pocket in more than oneconformation that is similar in overall binding energy. In those cases,the deformation energy of binding is taken to be the difference betweenthe energy of the free entity and the average energy of theconformations observed when the inhibitor binds to the protein.

An entity designed or selected as binding to any one of the abovebinding pockets may be further computationally optimized so that in itsbound state it would preferably lack repulsive electrostatic interactionwith the target enzyme and with the surrounding water molecules. Suchnon-complementary electrostatic interactions include repulsivecharge-charge, dipole-dipole and charge-dipole interactions.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interactions. Examples of programsdesigned for such uses include: Gaussian 94, revision C (M. J. Frisch,Gaussian, Inc., Pittsburgh, Pa. ©1995); AMBER, version 4.1 (P. A.Kollman, University of California at San Francisco, ©1995);QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. ©1998);Insight II/Discover (Molecular Simulations, Inc., San Diego, Calif.©1998); DelPhi (Molecular Simulations, Inc., San Diego, Calif. ©1998);and AMSOL (Quantum Chemistry Program Exchange, Indiana University).These programs may be implemented, for instance, using a SiliconGraphics workstation such as an Indigo2 with “IMPACT” graphics. Otherhardware systems and software packages will be known to those skilled inthe art.

Another approach enabled by this invention, is the computationalscreening of small molecule databases for chemical entities or compoundsthat can bind in whole, or in part, to any of the above binding pockets.In this screening, the quality of fit of such entities to the bindingpocket may be judged either by shape complementarity or by estimatedinteraction energy (E. C. Meng et al., J. Comp. Chem., 13, pp. 505-524(1992)).

Although the phosphorylated and unphosphorylated forms of GSK-3β havesimilar binding pockets, the subtle differences in water molecules,ions, position of the Y216 residue near the binding pockets as well asdeviations in the overall structure may render slightly differentresults in the calculation of binding energies for inhibitors. Bycomparing the binding energies of inhibitors to the phosphorylated andunphosphorylated form, one may select inhibitors that are more suitablefor one form than the other. Furthermore, the identification ofinhibitors for both forms would allow the options of inhibiting GSK-3βprior to or after phosphorylation by upstream kinases in vivo.

Another particularly useful drug design technique enabled by thisinvention is iterative drug design. Iterative drug design is a methodfor optimizing associations between a protein and a compound bydetermining and evaluating the three-dimensional structures ofsuccessive sets of protein/compound complexes.

In iterative drug design, crystals of a series of protein or proteincomplexes are obtained and then the three-dimensional structures of eachcrystal is solved. Such an approach provides insight into theassociation between the proteins and compounds of each complex. This isaccomplished by selecting compounds with inhibitory activity, obtainingcrystals of this new protein/compound complex, solving thethree-dimensional structure of the complex, and comparing theassociations between the new protein/compound complex and previouslysolved protein/compound complexes. By observing how changes in thecompound affect the protein/compound associations, these associationsmay be optimized.

In some cases, iterative drug design is carried out by formingsuccessive protein-compound complexes and then crystallizing each newcomplex. Alternatively, a pre-formed protein crystal is soaked in thepresence of an inhibitor, thereby forming a protein/compound complex andobviating the need to crystallize each individual protein/compoundcomplex. The phosphorylated crystals provided by this invention may besoaked in the presence of a compound or compounds, to provide othercrystal complexes.

Structure Determination of Other Molecules

The structure coordinates set forth in any one of FIGS. 1-7 can also beused to aid in obtaining structural information about anothercrystallized molecule or molecular complex. This may be achieved by anyof a number of well-known techniques, including molecular replacement.

According to an alternate embodiment, the machine-readable data storagemedium comprises a data storage material encoded with a first set ofmachine readable data which comprises the Fourier transform of at leasta portion of the structure coordinates set forth in any one of FIGS.1-7, and which, when using a machine programmed with instructions forusing said data, can be combined with a second set of machine readabledata comprising the X-ray diffraction pattern of a molecule or molecularcomplex to determine at least a portion of the structure coordinatescorresponding to the second set of machine readable data.

In another embodiment, the invention provides a computer for determiningat least a portion of the structure coordinates corresponding to X-raydiffraction data obtained from a molecule or molecular complex, whereinsaid computer comprises:

-   -   a) a machine-readable data storage medium comprising a data        storage material encoded with machine-readable data, wherein        said data comprises at least a portion of the structure        coordinates of GSK-3β according to any one of FIGS. 1-7;    -   b) a machine-readable data storage medium comprising a data        storage material encoded with machine-readable data, wherein        said data comprises X-ray diffraction data obtained from said        molecule or molecular complex; and    -   c) instructions for performing a Fourier transform of the        machine readable data of (a) and for processing said machine        readable data of (b) into structure coordinates.

For example, the Fourier transform of at least a portion of thestructure coordinates set forth in any one of FIGS. 1-7 may be used todetermine at least a portion of the structure coordinates of GSK-3βhomologues, and other sufficiently homologous kinases such as CDK2. Inone embodiment, the molecule is a GSK-3β homologue. In anotherembodiment, the molecular complex is selected from the group consistingof a GSK-3β protein complex and a GSK-3β homologue complex.

Therefore, in another embodiment this invention provides a method ofutilizing molecular replacement to obtain structural information about amolecule or molecular complex whose structure is unknown comprising thesteps of:

-   -   a) crystallizing said molecule or molecular complex of unknown        structure;    -   b) generating an X-ray diffraction pattern from said        crystallized molecule or molecular complex; and    -   c) applying at least a portion of the GSK-3β structure        coordinates set forth in any one of FIGS. 1-7 to the X-ray        diffraction pattern to generate a three-dimensional electron        density map of the molecule or molecular complex whose structure        is unknown.

By using molecular replacement, all or part of the structure coordinatesof the GSK-3β as provided by this invention (and set forth in any one ofFIGS. 1-7) can be used to determine the structure of a crystallizedmolecule or molecular complex whose structure is unknown more quicklyand efficiently than attempting to determine such information ab initio.

Molecular replacement provides an accurate estimation of the phases foran unknown structure. Phases are a factor in equations used to solvecrystal structures that can not be determined directly. Obtainingaccurate values for the phases, by methods other than molecularreplacement, is a time-consuming process that involves iterative cyclesof approximations and refinements and greatly hinders the solution ofcrystal structures. However, when the crystal structure of a proteincontaining at least a homologous portion has been solved, the phasesfrom the known structure provide a satisfactory estimate of the phasesfor the unknown structure.

Thus, this method involves generating a preliminary model of a moleculeor molecular complex whose structure coordinates are unknown, byorienting and positioning the relevant portion of the GSK-3β accordingto any one of FIGS. 1-7 within the unit cell of the crystal of theunknown molecule or molecular complex so as best to account for theobserved X-ray diffraction pattern of the crystal of the molecule ormolecular complex whose structure is unknown. Phases can then becalculated from this model and combined with the observed X-raydiffraction pattern amplitudes to generate an electron density map ofthe structure whose coordinates are unknown. This, in turn, can besubjected to any well-known model building and structure refinementtechniques to provide a final, accurate structure of the unknowncrystallized molecule or molecular complex (E. Lattman, “Use of theRotation and Translation Functions”, in Meth. Enzymol., 115, pp. 55-77(1985); M. G. Rossmann, ed., “The Molecular Replacement Method”, Int.Sci. Rev. Ser., No. 13, Gordon & Breach, New York (1972)).

The structure of any portion of any crystallized molecule or molecularcomplex that is sufficiently homologous to any portion of the GSK-3β canbe resolved by this method.

In a preferred embodiment, the method of molecular replacement isutilized to obtain structural information about a GSK-3 homologue. Thestructure coordinates of GSK-3β as provided by this invention areparticularly useful in solving the structure of GSK-3β complexes thatare bound by ligands, substrates and inhibitors.

Furthermore, the structure coordinates of GSK-3β as provided by thisinvention are useful in solving the structure of GSK-3β proteins thathave amino acid substitutions, additions and/or deletions (referred tocollectively as “GSK-3β mutants”, as compared to naturally occurringGSK-3β. These GSK-3β mutants may optionally be crystallized inco-complex with a chemical entity, such as a non-hydrolyzable ATPanalogue, a suicide substrate or a inhibitor. The crystal structures ofa series of such complexes may then be solved by molecular replacementand compared with that of wild-type GSK-3β. Potential sites formodification within the various binding pockets of the enzyme may thusbe identified. This information provides an additional tool fordetermining the most efficient binding interactions, for example,increased hydrophobic interactions, between GSK-3β and a chemical entityor compound.

The structure coordinates are also particularly useful to solving thestructure of crystals of GSK-3β or GSK-3β homologues co-complexed with avariety of chemical entities. This approach enables the determination ofthe optimal sites for interaction between chemical entities, includingcandidate GSK-3β inhibitors and GSK-3β. For example, high resolutionX-ray diffraction data collected from crystals exposed to differenttypes of solvent allows the determination of where each type of solventmolecule resides. Small molecules that bind tightly to those sites canthen be designed and synthesized and tested for their GSK-3β inhibitionactivity.

All of the complexes referred to above may be studied using well-knownX-ray diffraction techniques and may be refined versus 1.5-3.4 Åresolution X-ray data to an R value of about 0.30 or less using computersoftware, such as X-PLOR (Yale University, ©1992, distributed byMolecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth.Enzymol., vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press(1985)). This information may thus be used to optimize known GSK-3βinhibitors, and more importantly, to design new GSK-3β inhibitors.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

EXAMPLE 1 3-Amino-4,5-diphenyl-1H-pyrazolo[3,4-c]pyridazine (Compound 1)

The appropriate diaryl keto hydrazone (see Scheme I, formula 1, whereinX is H and Y is H; 0.85 mmol) and ethyl cyanoacetate (0.9 mmol) wereadded to 3 mL ethanol. Sodium ethoxide (0.9 mmol) in THF wassubsequently added and the mixture refluxed for 6 hours. After cooling,the solvent was removed under vacuum and the residue taken up in 10 mLdichloromethane. It was then washed with 0.1 M HCl, water and dried withsodium sulfate. After filtering, the solvent was removed under vacuumand the product, 4-cyano-5,6-diaryl 2(1H) pyridazinone (Scheme I,formula 2, wherein X is H and Y is H) was purified by chromatography onsilica gel (5:95 methanol/dichloromethane).

Purified 4-cyano-5,6-diaryl 2(1H) pyridazinone (100 mg) was added to 2mL POCl₃ and heated to 100° C. for 5-6 hours. After cooling, thereaction mixture was poured onto 10 mL ice and stirred for one hour. Theresulting 3-chloro-4-cyano-5,6-diaryl pyridazine (Scheme I, formula 3,wherein X is H and Y is H) was filtered off, washed with water, airdried and used in the next step without further purification.

Crude 3-chloro-4-cyano-5,6-diaryl pyridazine was refluxed with 2equivalents of anhydrous hydrazine in ethanol for several hours. Uponcooling, the product would sometimes precipitate out, in which case thepure title compound was obtained by recrystallizing from ethanol.Otherwise the title compound was purified by chromatography on silicagel (5:95 methanol-dichloromethane): MS (ES+) m/e: 288.01 (M+H);analytical HPLC(C₁₈ column) 2.96 minutes.

EXAMPLE 23-Amino-4(4-chlorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 2)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isH and Y is p-Cl): MS (ES+) m/e: 321.89 (M+H); analytical HPLC(C₁₈column) 3.33 minutes.

EXAMPLE 3 3-Amino-4,5-bis(4-fluorophenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 3)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isp-F and Y is p-F): MS (138+) m/e: 324.1 (M+H); analytical HPLC (C₁₈column) 3.26 minutes.

EXAMPLE 43-Amino-4-phenyl-5-(4-fluorophenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 4)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isp-F and Y is H): MS (ES+) m/e: 306.1 (M+H); analytical HPLC (C₁₈ column)3.08 minutes.

EXAMPLE 53-Amino-4-(4-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 5)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isH and Y is p-F): MS (ES+) m/e: 306.1 (M+H); analytical HPLC (C₁₈ column)3.02 minutes.

EXAMPLE 63-Amino-4-(3-fluorophenyl)-5-phenyl-1H-pyrazolo[3,4-c]pyridazine(Compound 6)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isH and Y is m-F): MS (ES+) m/e: 306.1 (M+H); analytical HPLC(C₁₈ column)2.94 minutes.

EXAMPLE 7 3-Amino-4-phenyl-5-(4-pyridyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 7)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate heteroaryl aryl ketohydrazone: MS (ES+) m/e: 289.1 (M+H); analytical HPLC (C₁₈ column) 1.77minutes.

EXAMPLE 83-Amino-4-phenyl-5-(3-fluorophenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 8)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X ism-F and Y is H): MS (ES+) m/e: 306.1 (M+H); analytical HPLC (C₁₈ column)3.12 minutes.

EXAMPLE 9 N-(4,5-Diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)-acetamide(Compound 9)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isH and Y is H), followed by amidation with CH₃CO₂H: MS (ES+) m/e: 330.1(M+H); analytical HPLC (C₁₈ column) 2.65 minutes.

EXAMPLE 10 N-(4,5-Diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-yl)-benzamide(Compound 10)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isH and Y is H), followed by amidation with benzoic acid: MS (ES+) m/e:414.2 (M+Na+); analytical HPLC (C₁₈ column) 2.90 minutes.

EXAMPLE 113-Amino-4-phenyl-5-(4-methyl-phenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 11)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isp-CH₃ and Y is H): MS (ES+) m/e: 302.1 (M+H); analytical HPLC (C₁₈column) 3.07 minutes.

EXAMPLE 123-Amino-4-phenyl-5-(2-methyl-phenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 12)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X iso-CH₃ and Y is H): MS (ES+) m/e: 302.1 (M+H); analytical HPLC (C₁₈column) 2.94 minutes.

EXAMPLE 133-Amino-4-phenyl-5-(3-methyl-phenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 13)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X ism-CH₃ and Y is H): MS (ES+) m/e: 302.1 (M+H); analytical HPLC (C₁₈column) 3.09 minutes.

EXAMPLE 143-Amino-4-phenyl-5-(2-chloro-phenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 14)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X iso-Cl and Y is H): MS (ES+) m/e: 322.1 (M+H); analytical HPLC (C₁₈column) 3.48 minutes.

EXAMPLE 153-Amino-4-phenyl-5-(2-fluorophenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 15)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isO—F and Y is H): MS (ES+) m/e: 306.1 (M+H); analytical HPLC (C₁₈ column)2.97 minutes.

EXAMPLE 163-Amino-4-phenyl-5-(4-chloro-phenyl)-1H-pyrazolo[3,4-c]pyridazine(Compound 16)

The title compound was obtained according to synthetic proceduresdescribed in EXAMPLE 1 using the appropriate diaryl keto hydrazone (X isp-Cl and Y is H): MS (ES+) m/e: 322 (M+H); analytical HPLC (C₁₈ column)4.06 minutes.

EXAMPLE 17 4-Phenyl-1H-pyrazolo[3,4-c]pyridazin-3-ylamine (Compound 32)

Step A: 3-Oxo-5-phenyl-2,3-dihydro-pyridazine-4-carbonitrile

Phenyl glyoxal (1.0 g, 7.46 mmol) and cyanoacetohydrazide (740 mg, 7.46mmol) were heated to reflux in 10 mL ethanol for 16 hours. Aftercooling, the solvent was evaporated and the crude brown mixture waspurified by silica chromatography (1:9 methanol/dichloromethane). Thestill impure product was further recrystallized from methanol affording70 mg pure product.

Step B: 3-Chloro-5-phenyl-pyridazine-4-carbonitrile

3-Oxo-5-phenyl-2,3-dihydro-pyridazine-4-carbonitrile (70 mg) wassuspended in 1 mL phosphorous oxychloride and heated to 100° C. for 6hours. The mixture was then cooled and poured onto ice. The resultingbrown solid product was filtered and air dried and used in the next stepwithout further purification.

Step C: 4-Phenyl-1H-pyrazolo[3,4-c]pyridazin-3-ylamine

3-Chloro-5-phenyl-pyridazine-4-carbonitrile obtained from Step B wassuspended in 1 mL ethanol with 23 μL hydrazine and the mixture wasrefluxed for several hours. The solvent was then evaporated and thetitle product was purified by silica gel chromatography (1:9methanol/dichloromethane): MS (ES+) 212 (M+H); HPLC 1.121 minutes.

EXAMPLE 18 K_(i) Determination for the Inhibition of GSK-3

Compounds were screened for their ability to inhibit GSK-3β (amino acids1-420) activity using a standard coupled enzyme system (Fox et al.(1998) Protein Sci. 7, 2249). Reactions were carried out in a solutioncontaining 100 mM HEPES (pH 7.5), 10 mM MgCl₂, 25 mM NaCl, 300 μM NADH,1 mM DTT and 1.5% DMSO. Final substrate concentrations in the assay were20 μM ATP (Sigma Chemicals, St Louis, Mo.) and 300 μM peptide(HSSPHQS(PO₃H₂)EDEEE (SEQ ID NO: 21), American Peptide, Sunnyvale,Calif.). Reactions were carried out at 30° C. and 20 nM GSK-3β. Finalconcentrations of the components of the coupled enzyme system were 2.5mM phosphoenolpyruvate, 300 μM NADH, 30 μg/ml pyruvate kinase and 10μg/ml lactate dehydrogenase.

An assay stock buffer solution was prepared containing all of thereagents listed above with the exception of ATP and the test compound ofinterest. The assay stock buffer solution (175 μl) was incubated in a 96well plate with 5 μl of the test compound of interest at finalconcentrations spanning 0.002 μM to 30 μM at 30° C. for 10 min.Typically, a 12 point titration was conducted by preparing serialdilutions (from 10 mM compound stocks) with DMSO of the test compoundsin daughter plates. The reaction was initiated by the addition of 20 μlof ATP (final concentration 20 μM). Reaction rates were obtained using aMolecular Devices Spectramax plate reader (Sunnyvale, Calif.) over 10min at 30° C. The K_(i) values were determined from the rate data as afunction of inhibitor concentration.

The GSK-3 inhibitory activity of certain compounds of this invention areshown in Table 11. For GSK-3 K_(i) values, “+++” represents ≦0.1 μM,“++” represents between 0.1 and 10 μM, and “+” represents ≧10 μM.

TABLE 11 Inhibitory Activity Compound No. K_(i) 1 +++ 2 + 3 ++ 4 + 5 +++6 +++ 7 + 8 +++ 9 ++ 10 ++ 11 +++ 12 ++ 13 ++ 14 +++ 15 +++ 16 ++ 32 ++

EXAMPLE 19 Expression and Purification of GSK-3β

Full length human GSK-3β (1-420) (SEQ ID NO: 1) with an N-terminalhexa-histidine tag (SEQ ID NO: 56) and a thrombin cleavage site wasoverexpressed in a baculo virus expression system. GSK-3β was purifiedusing Talon metal affinity chromatography (Clontech, Palo Alto, Calif.)followed by size-exclusion on a Superdex 200 column (Pharmacia, Uppsala,Sweden). The hexa-histidine tag (SEQ ID NO: 56) was then removed byincubation with thrombin (Calbiochem, La Jolla, Calif.). In addition tothe authentic thrombin site, a second cleavage product was identified 10amino acids downstream at Threonine 7. Incubation overnight at 4° C.with 12 U mg⁻¹ thrombin produced more than 90% GSK-3β (7-420), which wasused for crystallographic studies. The reaction was quenched with PMSFand thrombin was removed with benzamidine sepharose (Pharmacia, Uppsala,Sweden). To separate unphosphorylated GSK-3β (7-420) from thephosphorylated species and GSK-3β cleaved at the authentic thrombincleavage site, the protein was applied to a MonoS 10/10 column(Pharmacia, Uppsala, Sweden) equilibrated in 25 mM HEPES, pH 7.2, 10%Glycerol (v/v), 2 mM DTT. The protein was eluted with a linear gradientfrom 0 to 300 mM NaCl in 30 column volumes. Unphosphorylated GSK-3β(7-420) eluted at 150 mM NaCl. Phosphorylated GSK-3β (7-420) eluted ataround 200 mM NaCl The protein was dialyzed against 25 mM Tris pH 8.0containing 200 mM NaCl and 2 mM DTT at 4° C., concentrated to 15 mgml⁻¹, and centrifuged at 100,000×g prior to crystallization. All proteinmolecular weights were confirmed by electrospray mass spectrometry.Phosphorylation on Tyr 216 was confirmed by tryptic digest and MALDI-TOFspectrometry.

EXAMPLE 20 Crystallization of GSK-3β

Crystallization of GSK-3β was carried out using the hanging drop vapordiffusion technique. The unphosphorylated GSK-3β formed diamond shapecrystals over a reservoir containing 15% PEG 3350, 50 mM Na/KP04 pH 4.1,10 mM DTT. The crystallization droplet contained 1 μl of 15 mg ml⁻¹protein solution and 1 μl of reservoir solution. Crystals formed in lessthan 1 hour and were harvested in a reservoir solution after 12 hrs.

The phosphorylated form (Tyrosine 216) of GSK-3β formed plate-likecrystals over a reservoir containing Solution A (7-10% PEG 3350, I00 mMTris HCl pH 7.5, 5% Dimethylsulphoxide(DMSO)). The component DMSO wasimportant for the crystallaization of phosphorylated GSK-3β. Thecrystallization droplet contained 1 μl of protein (16 mg/mL) and 1 μl ofreservoir solution. The crystals formed overnight and were harvested inSolution A after a few days.

In order to obtain crystals of the ADP-peptide-GSK-3β complex, 0.3 mMprotein was mixed with 1.4 mM glycogen synthase peptide (residues 650 to661), 2 mM ADP and 2 mM MgCl. The mixture was incubated on ice for twohours. Small rod shaped crystals of the complex formed over a reservoircontaining 10-15% PEG 3350 and 50 mM ammonium fluoride. Crystals largeenough for data collection were obtained after repeated cycles of microseeding.

Once the above crystals were harvested in reservoir solution, they weretransferred to reservoir solutions containing increasing concentrationsof glycerol, starting with 2% and increasing to 5, 10, 15, 20, 25 and30%. After soaking the crystals in 30% glycerol for less than 5 minutes,the crystals were scooped up with a cryo-loop, frozen in liquid nitrogenand stored for data collection.

EXAMPLE 21 Formation of GSK-3β-Inhibitor Complex Crystals

Phosphorylated GSK-3β-inhibitor1 complex crystals were formed by soakingphosphorylated GSK-3β crystals in Solution A that also contained 500 μMof inhibitor 4,5-Diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-ylamine.

Crystals of unphosphorylated GSK-3β-inhibitor2-4 complexes were formedby co-crystallizing the protein with the inhibitors. The inhibitor wasadded to the concentrated GSK-3β protein solution right before settingup the crystallization drop. Alternatively, inhibitor could be added toa diluted protein solution, and the mixture concentrated to the requiredconcentration. The unphosphorylated GSK-3β protein co-crystallized withinhibitors(5-Methyl-2H-pyrazol-3-yl)-(2-pyridin-4-yl-quinazolin-4-yl)-amine,4-(5-Methyl-2-phenylamino-pyrimidin-4-yl)-1H-pyrrole-2-carboxylic acid(2-hydroxy-1-phenyl-ethyl)-amide), and(1H-Indazol-3-yl)-[2-(2-trifluoromethyl-phenyl)-quinazolin-4-yl]-amine)over a reservoir solution containing 15-20% PEG 3350, 0.1-1 M of KF,potassium formate or ammonium formate. The crystallization dropletcontained 1 μl of 10-20 mg/ml protein solution containing the inhibitorand 1 μl of reservoir solution.

Once the crystals of both forms were harvested, they were transferred toreservoir solutions containing increasing concentrations of glycerol,starting with 5% and increasing to 10, 15, 20, 25 and 30%. After soakingthe crystals in 30% glycerol for less than 5 minutes, the crystals werescooped up with a cryo-loop, frozen in liquid nitrogen and stored fordata collection.

EXAMPLE 22 X-Ray Data Collection and Structure Determination

X-ray diffraction data for the unphosphorylated GSK-3β, phosphorylatedGSK-3β, phosphorylated GSK-3β-inhibitor complex, unphosphorylatedGSK-3β-inhibitor complexes, phosphorylated GSK-3β-ADP-peptide complexstructures were collected on a Raxis 4 image plate, with mirror-focusedCuKa X-rays generated by a rotating-anode source. X-ray data used torefine the unphosphorylated GSK-3β structure was collected at beam line5.0.2 of the Advanced Light Source Lawrence Berkeley Laboratory,Berkeley, Calif. Data collected on the Raxis 4 image plate was processedwith DENZO and SCALEPACK (Otwinowski et al., Methods Enzymol., 180,51-62 (1989)) Data collected at ALS were processed with the programMOSFLM and the data was scaled using SCALA (Collaborative ComputationalProject, N., Acta Cryst., D50, pp. 760-763 (1994)).

The data statistics of the unphosphorylated form are summarized in Table12. The spacegroup of the unphosphorylated crystals was P2₁2₁2₁, withunit cell dimensions a=83 b=86 c=178 Å, α=β=γ=90°. The starting phasesfor unphosphorylated GSK-3β were obtained by molecular replacement usingcoordinates of CDK2 (Protein Data Bank Accession number 1AQ1) (Lawrie,A. M., et al., Nat. Struct. Biol., 4, pp. 796-801 (1997)) as a searchmodel in the program AMORE (Navaza, J. Acta. Cryst., 50, pp. 157-163(1994)). The asymmetric unit contained two molecules. Multiple rounds ofrebuilding with QUANTA and refinement with CNX resulted in a final modelthat included amino acid residues 25 to 384 for molecule A and residues37 to 382 for molecule B, 4 phosphate ions and 46 water molecules. The acarbons in A and B chains have a root-mean-squared deviation aftersuperposition of 0.48 Å. The refined model has a crystallographicR-factor of 23.7% and R-free of 27.4%. The coordinates of the structurehave been deposited with the Protein Databank (accession code I109).

The data statistics of the phosphorylated form are summarized in Table13. The spacegroup of the crystals was P1, with unit cell dimensionsa=64 Å b=67.2 Å c=67.4 Å α=100.1° β=103.5° γ=90° or a=64 Å b=67 Å c=67 Åα=80° β=77° γ=89.8°. The dimensions of the unit cell varied 1-2% fromcrystal to crystal. The starting phases for phosphorylated GSK-3β wereobtained by molecular replacement using coordinates of theunphosphorylated form as a search model in the program AMORE ENRfu,supra. The asymmetric unit contained two molecules. Multiple rounds ofrebuilding with QUANTA and refinement with CNX resulted in a final modelthat included residues 37 to 383 for molecule A and residues 37 to 383for molecule B and 83 water molecules. All data was included and NCSrestraint was applied through out the refinement. The final step ofrefinement was an individual B-factor refinement. The refined model hasa crystallographic R-factor of 23.8% and R-free of 27.6%.

The spacegroup of the unphosphorylated GSK-3β-inhibitor2-4 complexcrystals was P2₁2₁2₁, with unit cell dimensions a=83 b=86 c=178 Å,α=β=γ=90°. The starting phases for unphosphorylated GSK-3β-inhibitorcomplexes were obtained by molecular replacement using coordinates ofthe unphosphorylated form as a search model in the program AMORE ENRfu,supra. The asymmetric unit contained two molecules. Multiple rounds ofrebuilding with QUANTA and refinement with CNX were performed.

The data statistics of the unphosphorylated GSK-3β-inhibitor2 complexare summarized in Table 14. The structure was refined to 2.9 Å, and theR-factor was 24.1% and R-free was 28%. The final model included aminoacid residues 25 to 385 for molecule A, residues 37 to 382 for moleculeB, inhibitor2 and 6 water molecules.

For the unphosphorylated GSK-3β-inhibitor3 complex, the structure wasrefined to 2.3 Å, and the R-factor was 25.3% and R-free was 28.6%. Formolecule A, residues 25 to 381 were included in the final model. Formolecule B, amino acid residues 37 to 382 were included in the finalmodel.

For the unphosphorylated GSK-3β-inhibitor4 complex, the structure wasrefined to 2.8 Å, and the R-factor was 24.1% and R-free was 28.3%. Formolecule A, residues 36 to 381 were included in the final model. Formolecule B, amino acid residues 37 to 382 were included in the finalmodel.

The data statistics of the phosphorylated GSK-3β-inhibitor1 complex aresummarized in Table 15. The spacegroup of the crystals was P1, with unitcell dimensions a=64 Å b=67 Å c=67 Å a=100° β=103° γ=89.8° or a=64 Åb=67 Å c=67 Å α=80° β=77° γ=89.8°. The starting phases forphosphorylated GSK-3β-inhibitor complex were obtained by molecularreplacement using coordinates of the phosphorylated form as a searchmodel in the program AMORE ENRfu, supra. The asymmetric unit containedtwo molecules. Multiple rounds of rebuilding with QUANTA and refinementwith CNX resulted in a final model that included residues 37 to 383 formolecule A and residues 37 to 384 for molecule B, the inihibitors boundto molecule A and B, and 83 water molecules. The structure was refinedto 2.8 Å, and had an R-factor of 22.6% and R-free of 26.9%.

The data statistics of the phosphorylated GSK-3β-ADP-peptide complex aresummarized in Table 16. The spacegroup of the crystals was P2₁2₁2₁, withunit cell dimensions a=75.16 Å b=107.93 Å c=121.2 Å α=90° β90° γ=90°.The starting phases for phosphorylated GSK-3β were obtained by molecularreplacement using coordinates of the unphosphorylated form as a searchmodel in the program AMORE ENRfu, supra. The asymmetric unit containedtwo molecules. The glycine rich loop was well ordered and could be builtin the model. Multiple rounds of rebuilding with QUANTA and refinementwith CNX was performed. All data was included and NCS restraint wasapplied through out the refinement. The final step of refinement was agrouped B-factor refinement. The refined model has a crystallographicR-factor of 23.5% and R-free of 27.2%.

The Ramachandran plot of phosphorylated GSK-3β in complex with ADP andglycogen synthase peptide showed that 80.1% of the residues were in mostfavored regions and 19.6% in additionally allowed regions. TheRamachandran plot of apo-phosphorylated GSK-3β showed that 85.3% of theamino acid residues were in most favored regions and 14.3% inadditionally allowed regions. Two amino acid residues (Cys 218 inmolecule A and B) in both crystal structures were in disallowed regions.

In the above models, disordered residues were not included in the model.Alanine or glycine residues were used in the model if the side chains ofcertain residues could not be located in the electron density.

EXAMPLE 23 Overall Structure of Unphosphorylated GSK-3β

GSK-3β has the typical 2 domain kinase fold (Hanks, S. K., et al.,Science, 241, pp. 42-52 (1988); Hanks, S. K. and A. M. Quinn, MethodsEnzymol., 200, pp. 38-62 (1991)) with a β-strand domain (amino acidresidues 25-138) at the N-terminal end and an α-helical domain at theC-terminal end (amino acid residues 139-343) (FIG. 8). The active siteis at the interface of the α-helical and β-strand domain, and isbordered by the glycine rich loop and the hinge. The activation loopruns along the surface of the substrate-binding groove. The C-terminal39 amino acid residues (amino acid residues 344-382) are outside thecore kinase fold and form a small domain that packs against theα-helical domain. The β-strand domain consists of seven anti-parallelβ-strands. Strands 2 to 6 form a β-barrel that is interrupted betweenstrand 4 and 5 by a short helix (amino acid residues 96-102) which packsagainst the β-barrel. This helix is conserved in all kinases, and two ofits residues play key roles in the catalytic activity of the enzyme.Arginine 96 is involved in the alignment of the two domains. Glutamate97 is positioned in the active site and forms a salt bridge with lysine85, a key residue in catalysis.

Phosphorylation of Peptides by GSK-3β

Before a serine/threonine kinase can phosphorylate a substrate, itsβ-strand and α-helical domains must be aligned into a catalyticallyactive conformation. Most kinases use one or two phosphorylated aminoacid residues on the activation loop for this purpose. Polar amino acidresidues, typically arginines and lysines, from the β-strand andα-helical domains, bind the phosphate group of the phosphorylated aminoacid residue on the activation loop, which leads to proper alignment ofthe two domains. The second phosphorylated amino acid residue (ifpresent, for example Tyr216 in GSK-3β) opens the substrate-bindinggroove and allows the substrate to bind.

Comparison of the GSK-3β with other kinases such as CDK2, p38γ and ERK2revealed that the structure of apo-GSK-3β resembles closely thesubstrate-bound, activated form of a kinase. The activation loop (aminoacid residues 200 to 226) in the GSK-3β structure is well ordered, andis positioned against the α-helical domain. This orientation opens thepeptide substrate binding groove (FIG. 11), and mimics the position ofthe activation loop of the activated substrate bound CDK2 (FIG. 10) butnot apo-CDK2 (Protein Data Bank Accession number 1HCL) (Schulze-Gahmen,U., et al., Proteins, 22, pp. 378-91 (1995)). Comparison of theactivation loops of GSK-3β, P38γ (Protein Data Bank Accession number1CM8) (Bellon, S., et al., Structure Fold Des, 7, pp. 1057-65 (1999)),substrate-bound activated CDK2 (Protein Data Bank Acession number 1QMZ)(Brown, N. R., et al., Nat. Cell. Biol., 1, pp. 438-443 (1999)), andERK2 (Protein Data Bank Accession number 2ERK) (Canagarajah, B. J., etal., Cell, 90, pp. 859-69 (1997)) shows that the backbone and side chainatoms of important residues align (FIG. 10). R96, R180 and K205 ofGSK-3β (FIG. 10A) superimpose well with R73, R152 and R176 ofphosphorylated P38y, respectively (FIG. 10C). These amino acid residuesalso superimpose well with the corresponding residues in phosphorylatedERK2 and substrate bound activated CDK2. In p38γ, CDK2 and ERK2, theseresidues point to the phosphate group from the phosphorylated threonineon the activation loop, the residue that is important for aligning theN-terminal and C-terminal domains. In the GSK-3β structure, R96, R180and K205 point to a PO₄ ⁻ ion that is located in the same position asthe phosphate group of the phosphorylated threonine in CDK2, ERK2 andp38γ.

The superposition of the GSK-3β and p38γ activation loops shows that theGSK-3β phosphorylation site, Y216, is located in a similar position asthe phospho-tyrosine (amino acid residue 185) of p38γ. Thephospho-tyrosine of p38γ acts as a gatekeeper for the substrate-bindinggroove. When it is phosphorylated, its side chain moves out of thegroove allowing substrate peptides to bind. The side chain of Y216 ofGSK-3β is also positioned to block access to the substrate-bindinggroove.

The sequence of GSK-3β is 25% identical and 41% similar to the sequenceof CDK2. The structure of GSK-3β presented here superimposes well withthe structure of activated, substrate-bound CDK2 (FIG. 9). Because ofthe similarity in sequence and fold, we can use the structure ofactivated, substrate-bound CDK2 as a model for the substrate binding ofGSK-3β.

Primed Phosphorylation

The GSK-3β substrate-binding groove is partially occupied by a loop froma neighboring GSK-3β molecule in the crystal (FIG. 11A). The loop ispositioned in front of the active site. Superposition of the α-helicaldomains of activated substrate-bound CDK2 and GSK-3β shows that fourresidues in the loop, DSGV (amino acid residues 260 to 263), are in avery similar position as the peptide in activated substrate-bound CDK2.S261 of the loop in GSK-3β occupies the same position as the targetserine in peptide-bound CDK2 (compare position S261 in FIG. 11A with S*in FIG. 11B). The CDK2-bound peptide has an extended conformation, whileloop 260-264 in the GSK-3β adopts a turn and occupies a small portion ofthe substrate-binding groove. It is likely that the natural substratefor GSK-3β also has an extended conformation, similar to the peptidebound to CDK2. If we use the CDK2-bound peptide as a model for a GSK-3βsubstrate, it becomes clear why GSK-3β prefers a phosphorylated serineor threonine at the P+4 position. The phosphate group at the P+4position will occupy the same position as the phosphate ion near theactivation loop of our structure, contacting R96, R180 and K205 (FIG.11). This means that while CDK2, p38γ and ERK2 use a phospho-threonineon the activation loop to align the β-strand and α-helical domains,GSK-3β uses the phosphorylated serine at the P+4 position of thesubstrate to align the two domains for optimal catalytic activity.

EXAMPLE 24 Active Site of GSK-3β-Inhibitor Complexes

Inhibitor1 4,5-Diphenyl-1H-pyrazolo[3,4-c]pyridazin-3-ylamine is boundin the deep cleft of the active site in the phosphorylated GSK-3βstructure (FIG. 14). Inhibitorl forms two hydrogen bonds with the hingeportion of the active site. The 1H pyrazole nitrogen shares a protonwith the D133 backbone carbonyl. The other pyrazole nitrogen (position2) accepts a proton from the V135 backbone nitrogen. The side chains ofL132 and K85 are positioned inside the active site. K85 is acatalytically important residue and forms a salt bridge with E97 (notshown).

Inhibitor2(5-Methyl-2H-pyrazol-3-yl)-(2-pyridin-4-yl-quinazolin-4-yl)-amine isbound in the active site in the unphosphorylated GSK-3β structure (FIG.13). The inhibitor2 forms four H-bonds with the hinge backbone. Twohydrogen bonds come from the pyrazole ring. The nitrogen in position onedonates a hydrogen to the backbone carbonyl of Asp 133. The nitrogen inposition 2 accepts a hydrogen from the Val 135 amide nitrogen. Thebackbone carbonyl of Val 135 is within hydrogen bonding range ofhydrogen donating groups on the inhibitor2. It contacts the linkernitrogen and the quinazoline carbon at position 8.

Inhibitor34-(5-Methyl-2-phenylamino-pyrimidin-4-yl)-1H-pyrrole-2-carboxylic acid(2-hydroxy-1-phenyl-ethyl)-amide) is bound in the active site in theunphosphorylated GSK-3β structure (FIG. 15). Inhibitor3 is a potentinhibitor of GSK-3β with a Ki of 4 nM. Inhibitor3 forms 5 hydrogen bondswith the GSK-3β protein. The three amino-pyrimidine hydrogen bondscontact the hinge backbone. The carbonyl forms a hydrogen bond with theside chain of the catalytic lysine (K85) and the hydroxyl forms ahydrogen bond with the side chain of asparagine 186. Asn186 is aconserved residue in GSK-3β and ERK2. The 5-methyl group of theamino-pyrimidine ring points toward L132 and V110 in GSK-3β. There islimited space between these two residues suggesting that a smallsubstituent is allowed in this position.

Inhibitor4(1H-Indazol-3-yl)-[2-(2-trifluoromethyl-phenyl)-quinazolin-4-yl]-amine)is bound in the active site in the unphosphorylated GSK-3β structure(FIG. 16). The indazole ring of inhibitor4 forms two hydrogen bonds withthe hinge backbone. The nitrogen in position one donates a hydrogen tothe backbone carbonyl of Asp 133. The nitrogen in position 2 accepts ahydrogen from the Val 135 amide nitrogen. The backbone carbonyl of Val135 is within hydrogen bonding range of hydrogen donating groups frominhibitor4. It contacts the linker nitrogen and the quinazoline carbonat position 8. The trifluoromethyl phenyl ring is almost perpendicularto the quinazoline ring with the ortho trifluoromethyl substituentpointing to the glycine rich loop. The side chain of glutamine 185 packsagainst the trifluoromethyl phenyl ring and points to the glycine richloop.

EXAMPLE 25 Overall Structure of apo-Phosphorylated GSK-3β andSubstrate-bound Phosphorylated GSK-3β

The apo-phosphorylated GSK-3β and substrate-bound phosphorylated GSK-3βstructures have the typical two-domain kinase fold (residues 37 to 343)(Hanks, S. K. and Quinn, A. M., Methods Enzymol., 200, pp. 38-62 (1991);Hanks, S. K. and Hunter, T., FASEB J., 9, pp. 576-96 (1995)). TheN-terminal β-strand domain (amino acid residues 37 to 138) forms aP-barrel consisting of seven β-strands (FIG. 17). The C-terminalα-helical domain contains amino acid residues 139 to 343. The C-terminal40 amino acid residues (residues 344 to 383) are not part of the kinasefold, but pack against the α-helical domain.

The active site and the substrate-binding groove are located at theinterface of the β-strand and α-helical domain. The active site isbordered by the hinge and the glycine-rich loop and contains anADP-molecule. The substrate-binding groove contains a 12 amino acidresidue phosphorylated peptide derived from the sequence in glycogensynthase recognized by GSK-3β (650 HSSPHQpSEDEEE 661 (SEQ ID NO: 21)).The peptide is positioned between the activation loop (amino acidresidues 200 to 226) and the β-strand domain (FIG. 17).

Comparison between the structures of unphosphorylated, phosphorylatedapo-GSK-3β and phosphorylated peptide-bound GSK-3β reveal localdifferences induced by the presence of the substrates (FIG. 18).Superposition of the protein backbone of unphosphorylated andphosphorylated apo-GSK-3β resulted in a mean displacement of 0.7 Å and amaximum displacement of 3.7 Å of the Ile 217 backbone carbonyl. In thephosphorylated peptide-bound GSK-3β structure, the phosphorylated sidechain of Tyr 216 rotated out of the substrate-binding groove and induceda 180° flip of the Ile 217 backbone carbonyl. As a consequence of thisadjustment, the torsion angles of Cys 218 appeared in disallowed regionsof the Ramachandran plot. In the phosphorylated apo-GSK-3β structure,the side chain of Y216 also flips out of the substrate binding groove(FIG. 10). The N-terminal domain of the phosphorylated peptide-boundGSK-3β rotated by 6.5°, with the glycine rich loop covering the ADPmolecule. The reorganization of the glycine rich loop resulted in 4.1 Åtranslation of the Ser 66 α-carbon. The loop connecting β-3 to α-C (L4,amino acid residues 87 to 95) migrated 13 Å (amino acid residue 92)towards the substrate-binding groove, and form contacts with thebackbone atoms of the glycogen synthase peptide. The αG helix (aminoacid residues 262 to 273) rotated towards phosphorylated Tyr 216. Therotation of the β-strand domain and the adjustments of the glycine richloop and L4 were induced by the presence of ADP and the substratepeptide since the β-strand domain of apo-phosphorylated GSK-3β were notrotated in comparison to unphosphorylated GSK-3β.

The loop connecting α1L14 with Trp 301 (amino acid residues 284 to 300)was poorly ordered in the apo- and ADP, peptide-bound structures. Thisloop is part of the Fratl binding site (Bax, B. et al., Structure(Camb),9, pp. 1143-52 (2001)) and covers a hydrophobic groove between αG andamino acid residues 288-294. The loop is separate from thesubstrate-binding groove and does not seem to influence peptidephosphorylation even when Frat1 is bound to GSK-3β (Thomas, G. M. etal., FEBS Lett., 458, pp. 247-51 (1999)).

EXAMPLE 26 The Active site of the GSK-30-ADP-peptide Complex

The active site is located at the interface of the β-strand andα-helical domain and is enclosed by the glycine rich loop and the hinge.The glycine rich loop is formed by two anti-parallel β-strands. Theconformation of the glycine rich loop adjusts to the ligand thatoccupies the active site. The absence of the γ-phosphate in the ADPmolecule allows the glycine rich loop to descend closer to the α-helicaldomain. A similar adjustment of the glycine rich loop was observed inthe PKA-ADP complex (Protein Data Bank Accession number 1JBP)(Madhusudan, Trafny, E. A. et al., Protein Sci, 3, pp. 176-87 (1994)).

The entire β-strand domain rotated 6.5° as a result of the ADP moleculebeing bound in the active site. The adenine moiety of ADP is surroundedby hydrophobic amino acid residues from the glycine rich loop (Ile 62,Val 70), from the hinge (Tyr 134) and from the bottom of the active site(Leu 188, Cys 199) (FIG. 19A). Two hydrogen bonds between the base andthe hinge backbone were observed. The amino group on position 6 forms ahydrogen bond with the backbone carbonyl of Asp 133, and the N1 nitrogenaccepts a hydrogen from Val135 amide nitrogen. The 3′ hydroxyl from theADP ribose donates a hydrogen to the backbone carbonyl of Gln 185. Theactive site residues that play a role in the serine phosphorylationreaction form a web of hydrogen bonds around the ADP phosphates and theglycogen synthase target serine (FIG. 19B). The two ADP phosphates forma complex with one Mg ion, which in turn contacts the side chains of Asp200 and Gln 185. Asp 200 is part of the Asp Phe Gly motif and is thefirst amino acid residue of the activation loop. The Asp 200 homologuein other kinase-AMPPNP complexes binds a second Mg ion (or Mn ion) thatis positioned between the β- and γ-phosphate (Bellon, S. et al.,Structure Fold Des., 7, pp. 1057-65 (1999); Xie, X. et al., Structure,6, pp. 983-91 (1998); Bossemeyer, D. et al., EMBO J, 12, pp. 849-59(1993)). However, a second Mg ion could not be located in our electrondensity maps. On the other hand, the structure of the PKA-ADP complex(Protein Data Bank Accession number 1JBP) (Madhusudan, Trafny, E. A. etal., Protein Sci, 3, pp. 176-87 (1994)) does not have any Mg ionassociated with the phosphate groups.

Lys 85 is positioned between Glu97 and the α- and β-phosphates andlikely facilitates the transfer of the γ-phosphate from ATP to thesubstrate serine. Asp 181 is also a conserved residue in kinases and itsside chain is within hydrogen bonding range of the peptide target serine(Ser 652). The backbone carbonyl of Asp 181 forms a hydrogen bond withthe Gln 185 side chain. Asp 181 prepares the serine hydroxyl for thenucleophilic attack on the γ-phosphate (Adams, J. A., Chem. Rev., 101,pp. 2271-2290 (2001)). Lys 183 makes a hydrogen bond with the peptidetarget serine. Lys 183 is positioned between the phosphate groups andthe target serine and is involved in the phosphate transfer. When theγ-phosphate is present in the kinase active site, the Lys 183 is likelyto contact the terminal phosphate group.

EXAMPLE 27 The Substrate-Binding Groove of the GSK-3β-ADP-PeptideComplex

GSK-3β has one phosphorylation site in the activation loop, Y216, whichacts as a gate for the substrate-binding groove. In the crystalstructure of unphosphorylated GSK-3β, the unphosphorylated tyrosyl sidechain occupied the substrate-binding groove. A comparison between theunphosphorylated and phosphorylated peptide-bound GSK-3β structuresshowed that there would be space to accommodate the glycogen synthasepeptide even if the unphosphorylated tyrosine remains in thesubstrate-binding groove. The P+2 residue of the peptide (H is 654) isthe closest amino acid residue to Tyr 216. Its imidazole ring is part ofa cluster of aromatic residues formed by GSK-3β residues Phe 67, Phe 93and the peptide substrate amino acid residue H is 650.

The phosphorylated Tyr216 side chain has moved out of thesubstrate-binding groove in the GSK-3β-ADP-peptide complex structure(FIG. 21). The phospho-phenol group of Tyr 216 is bound to the sidechains of Arg 220 and Arg 223, which results in neutralization of thenegative charge of the phosphate group and a 180° flip of the Ile 217backbone carbonyl. The phospho-phenol group also caused a slightadjustment of the αG helix (residues 262-272), which results in thedistance between the phosphate oxygen atoms and amide nitrogen of G262(which is the closest H-bond donor) outside the H-bonding range (4.4 Å).

Glycogen synthase is a major substrate of GSK-3β with multiplephosphorylation sites. The canonical phosphorylation site for GSK-3β isSXXXpS. The P+4 serine (or threonine) is first phosphorylated by adifferent kinase, which is called primed phosphorylation. In the case ofglycogen synthase, GSK-3β phosphorylates four sites sequentially afterit is phosphorylated by casein kinase-II. The co-crystallized peptidepresent in the substrate-binding groove is derived from glycogensynthase. The sequence (650-HSSPHQ(pS)EDEEE-661 (SEQ ID NO: 21))contains the canonical GSK-3β phosphorylation motif and GSK-3βcanphosphorylate Ser 652 under the appropriate conditions. Ten of thetwelve residues (residues 650 to 659) had visible electron density andwere built in the substrate-binding groove (FIG. 21). The peptide hasthe shape of a large loop with residues 651 to 656 fitting in thesubstrate binding groove and residues 650, 657 to 659 exposed tosolvent. The structure reveals why GSK-3 β prefers a phosphorylatedserine or threonine at the P+4 position of the glycogen synthasephosphorylation site. The phosphate group of Ser 656 occupies apositively surface charged pocket formed by residues Arg 96, Arg 180 andLys 205. These amino acid residues are conserved in serine/threoninekinases and are responsible for the proper alignment of the β-strand andα-helical domains, the latter of which is required for optimal catalyticactivity. Other serine/threonine kinases such as CDK2 (Schulze- Gahmen,U. et al., Proteins, 22, pp. 378-91 (1995)), ERK2 (Zhang, F. et al.,Nature, 367, pp. 704-11 (1994); Canagarajah, B. J. et al., Cell, 90, pp.859-69 (1997)) and p38γ (Bellon, S. et al., Structure Fold Des., 7, pp.1057-65 (1999)) have a phosphorylated threonine in the activation loopfor this purpose, but GSK-3 uses a phosphorylated residue from thesubstrate. The target serine (Ser 652) is positioned in front of theactive site, 6.0 Å from the β-phosphate of the ADP molecule. The loopbetween residues Lys 85 and Arg 96 migrates more than 10 Å to facilitatethe glycogen synthase peptide binding. Phe 93 forms a pi-stack with thepeptide His 654. Ser 66, which is at the tip of the glycine rich loop,makes a hydrogen bond with the target serine backbone-nitrogen (Ser652). The backbone carbonyl of pSer 656 forms a hydrogen bond with Lys94.

The canonical phosphorylation motif for GSK-3β is SXXXpS. There is nosequence requirement for the three residues between the target serineand the phosphoserine. The P+1 residue in the glycogen synthase peptideused here is a proline. Although prolines are not uncommon as the P+1residue in phosphorylation motifs, the GSK-3β phosphorylation does notrequire a proline. A sequence analysis of the phosphorylation motifs ofthe proteins that undergo primed phosphorylation shows that residuessuch as A, G, Q, E, S, T, R and V can replace the proline at the P+1position. In the GSK-3β-ADP-peptide structure, the proline side chainfits in a pocket formed by the side chains of pY216, R220, R223 and thebackbones of residues 217 to 219. The pocket is shallow, and thedistance between the Cγ of Pro 654 and Arg 223 is 3.8 Å. This pocket canonly accommodate small residues such as Ala, Ser or Val. Largerresidues, such as Arg and Gln will have to point their side chains intothe solvent. This might explain the absence of bulky hydrophobicresidues at the P+1 position. This fact is reflected in the interactionsbetween the glycogen synthase peptide and GSK-3β. Except for thephosphoserine side chain interactions with the basic residues, the otherinteractions are with the peptide backbone. The pi-stack interactionbetween H is 654 and Phe 93 is probably specific for the phosphorylationof Ser 652, because there is no aromatic residue at the P+2 position inother motifs of glycogen synthase or eIF2b.

The crystal structure of the GSK-3β-ADP-peptide complex provides adetailed picture of the phosphorylation of the glycogen synthasepeptide. Although the ADP molecule does not contain the γ-phosphate, thecatalytic residues are located around the ADP molecule similar to thepositions in other kinase nucleotide complexes. The phosphoserine of thepeptide serves as an anchor for the peptide and is required for theproper alignment of the β-strand and α-helical domains of GSK-3β.

Biological Implications

Activation of the insulin-signaling pathway induces increased glucoseuptake and conversion to glycogen. Patients with type II diabetes havedecreased sensitivity towards insulin, which reduces glycogen synthesisand increased blood glucose levels. The conversion of glucose intoglycogen by glycogen synthase is the rate limiting step in glycogensynthesis and the phosphorylation status of glycogen synthase determinesthe catalytic rate. Glycogen synthase has at least nine phosphorylationsites and the more it is phosphorylated the lower its catalyticactivity. GSK-3β is one of the kinases that phosphorylates and inhibitsglycogen synthase. It phosphorylates sequentially multiple sites at theC-terminal end of glycogen synthase after Casein Kinase IIphosphorylates glycogen synthase. The canonical sequence recognized byGSK-3β is SXXXpS, which is four times present in glycogen synthase.GSK-3β is a potential therapeutic target for type 2 diabetes because itsinhibition leads to increased glycogen synthesis and decreased bloodglucose levels.

The crystal structures of apo-phosphorylated GSK-3β and GSK-3β incomplex with ADP and glycogen synthase peptide provide insight on howGSK-3β phosphorylates its substrates. The ADP molecule occupies theactive site and the glycogen synthase peptide occupies thesubstrate-binding groove. The phosphorylated serine at the P+4 positionof the glycogen synthase peptide binds three well-conserved basicresidues, which results in optimal alignment of the β-strand andα-helical domains of the GSK-3β kinase core. Other interactions betweenGSK-3β and the glycogen synthase peptide involve mostly backbone atomsof the peptide, which might explain the tolerance for different residuesat position P+1, P+2 and P+3. The present invention will be helpful inunderstanding the role of GSK-3β in the insulin-signaling pathway anddevelopment of potential new anti-diabetic therapies.

EXAMPLE 28 The Use of GSK-3β Coordinates for Inhibitor Design

The coordinates of any one of FIGS. 1-7 are used to design compounds,including inhibitory compounds, that associate with GSK-3β or GSK-3βhomologues. This process may be aided by using a computer comprising amachine-readable data storage medium encoded with a set ofmachine-executable instructions, wherein the recorded instructions arecapable of displaying a three-dimensional representation of the GSK-3β,the GSK-3β homologues or portions thereof. The graphical representationis used according to the methods described herein to design compounds.Such compounds associate with the GSK-3β or GSK-3β homologue at theactive site or substrate binding pocket.

While we have described a number of embodiments of this invention, it isapparent that our basic constructions may be altered to provide otherembodiments which utilize the products, processes and methods of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims, rather than by thespecific embodiments which have been presented by way of example.

U.S. provisional applications 60/287,366, 60/361,899, 60/297,094 areincorporated herein by reference in their entirety.

TABLE 12 Summary of data collection Source R-Axis IV ALS 5.0.2Wavelength (Å) 1.54 1.1 Resolution (Å) 3.0  2.7 No. of Reflections303687/26039 251279/34993 (measured/unique) Completeness (%)  96.7/89.3 98.9/99.8 (overall/outer shell) R_(merge) (%)¹ 0.064/0.30 0.070/0.32(overall/outer shell) Structure refinement Resolution (Å) 48.3-2.7 No.of reflections 34747 R factor 23.7 Free R factor† 27.4 Rms deviationsBond lengths 0.01 Bond angles 1.5° ¹R_(merge) = 100 × Σ_(h)Σ_(i)|I_(hi)− <I_(h)>|/Σ_(h)Σ_(i)I_(hi). †The Free R factor was calculated with 9.1%of the data.

TABLE 13 Summary of data collection Source R-Axis IV Wavelength (Å) 1.54Resolution (Å) 2.5 No. of Reflections 32357/3447 (measured/unique)Completeness (%)  87.0/61.8 (overall/outer shell) I/δ (I) 10.4/2.3(overall/outer shell) R_(merge) (%) 0.064/0.30 (overall/outer shell)Structure refinement Resolution (Å) 42.8-2.5 No. of reflections 32357 Rfactor 23.8 Free R factor¹ 27.6 Rms deviations Bond lengths 0.008 Bondangles 1.5° Bfactor (average)² 45 Å ¹The Free R factor was calculatedwith 10% of the data. ²The B-factor of the data (Wilson plot) was 26.4Å².

TABLE 14 Summary of data collection Source R-Axis IV Wavelength (Å) 1.54Resolution (Å) 2.9 No. of Reflections 338559/26836 (measured/unique)Completeness (%)  93.1/85.1 (overall/outer shell) I/δ (I) 10.2/2.7(overall/outer shell) R_(merge) (%) 0.072/0.29 (overall/outer shell)Structure refinement Resolution (Å) 25.7-2.9 No. of reflections 26796 Rfactor 24.0 Free R factor¹ 27.9 Rms deviations Bond lengths 0.011 Bondangles 1.8° Bfactor (average)² 39.3 Å ¹The Free R factor was calculatedwith 9.4% of the data. ²The B-factor of the data (Wilson plot) was 33Å².

TABLE 15 Summary of data collection Source R-Axis IV Wavelength (Å) 1.54Resolution (Å) 2.8 No. of Reflections  71493/23161 (measured/unique)Completeness (%)  88.3/90.3 (overall/outer shell) I/δ (I) 14.7/2.2(overall/outer shell) R_(merge) (%) 0.046/0.33 (overall/outer shell)Structure refinement Resolution (Å) 32.2-2.8 No. of reflections 23148 Rfactor 22.5 Free R factor¹ 26.8 Rms deviations Bond lengths 0.013 Bondangles 1.6° Bfactor (average)² 51.1 Å ¹The Free R factor was calculatedwith 9.4% of the data. ²The B-factor of the data (Wilson plot) was 91.3Å².

TABLE 16 Summary of data collection Source R-Axis IV Wavelength (Å) 1.54Resolution (Å) 2.8 No. of Reflections 311458/24709 (measured/unique)Completeness (%)  99.4/99.8 (overall/outer shell) I/δ (I) 18.4/3.6(overall/outer shell) R_(merge) (%) 0.075/0.33 (overall/outer shell)Structure refinement Resolution (Å) 49.3-2.8 No. of reflections 24288 Rfactor 23.5 Free R factor¹ 27.2 Rms deviations Bond lengths 0.013 Bondangles 1.7° ¹The Free R factor was calculated with 9.4% of the data.

1. A crystal comprising an unphosphorylated Glycogen Synthase Kinase-3β(GSK-3β) protein complexed to a chemical entity, wherein said GSK-3βprotein consists of amino acids 7-420 of SEQ ID NO:1; wherein saidcrystal is in space group P2₁2₁2₁, and has unit cell dimensions ofa=83±2 Å, b=86±2 Å, c=178±2 Å, α=β=γ=90°; and wherein said chemicalentity is selected from the group consisting of:(5-Methyl-2H-pyrazol-3-yl)-(2-pyridin-4-yl-quinazolin-4-yl)-amine;4-(5-Methyl-2-phenylamino-pyrimidin-4-yl)-1H-pyrrole-2-carboxylic acid(2-hydroxy-1-phenyl-ethyl)-amide; and(1H-Indazol-3-yl)-[2-(2-trifluoromethyl-phenyl)-quinazolin-4-yl]-amine).2. A method of obtaining a crystal comprising an unphosphorylatedGlycogen Synthase Kinase-3β (GSK-3β) protein complexed to a chemicalentity, wherein said GSK-3β protein consists of amino acids 7-420 of SEQID NO:1, and wherein said crystal is in space group P2₁2₁2₁, and hasunit cell dimensions of a=83±2 Å, b=86±2 Å, c=178±2 Å, α=γ=90°;comprising the steps of: a) producing and purifying said GSK-3β protein;b) forming a protein complex by contacting the purified GSK-3β proteinof step a) with a chemical entity is selected from the group consistingof: (5-ethyl-2H-pyrazol-3-yl)-(2-pyridin-4-yl-quinazolin-4-yl)-amine;4-(5-Methyl-2-phenylamino-pyrimidin-4-yl)-1H-pyrrole-2-carboxylic acid(2-hydroxy-1-phenyl-ethyl)-amide; and(1H-Indazol-3-yl)-[2-(2-trifluoromethyl-phenyl)-quinazolin-4-yl]-amine);c) mixing a crystallization solution with the protein complex of step b)to produce a crystallizable composition; and d) subjecting thecrystallizable composition of step c) to conditions which promotecrystallization, thereby obtaining said crystal.
 3. The method of claim1, wherein the GSK-3β protein is produced from a baculovirusoverexpression system.